Therapy and diagnosis of conditions related to telomere length and/or telomerase activity

ABSTRACT

Method and compositions are provided for the determination of telomere length and telomerase activity, as well as the ability to inhibit telomerase activity in the treatment of proliferative diseases. Particularly, primers are elongated under conditions which minimize interference from other genomic sequences, so as to obtain accurate determinations of telomeric length or telomerase activity. In addition, compositions are provided for intracellular inhibition of telomerase activity and means are shown for slowing the loss of telomeric repeats in aging cells.

[0001] This application is a continuation-in-part of Michael D. West etal., entitled “Therapy and diagnosis of conditions related to telomerelength and/or telomerase activity, filed Mar. 24, 1993, and assignedU.S. Ser. No. 08/038,766, which is a continuation-in-part of Michael D.West et al., entitled “Telomerase Activity Modulation and TelomereDiagnosis”, filed May 13, 1992, and assigned U.S. Ser. No. 07/882,438,both (including drawings) hereby incorporated by reference herein.

[0002] This invention relates to methods for therapy and diagnosis ofcellular senescence and immortalization.

BACKGROUND OF THE INVENTION

[0003] The following is a general description of art relevant to thepresent invention. None is admitted to be prior art to the invention.Generally, this art relates to observations relating to cellularsenescence, and theories or hypothesis which explain such aging and themechanisms by which cells escape senescence and immortalize.

[0004] Normal human somatic cells (e.g., fibroblasts, endothelial, andepithelial cells) display a finite replicative capacity of 50-100population doubling characterized by a cessation of proliferation inspite of the presence of abundant growth factors. This cessation ofreplication in vitro is variously referred to as cellular senescence orcellular aging, See, Goldstein, 249 Science 1129, 1990; Hayflick andMoorehead, 25 Exp. Cell Res. 585, 1961; Hayflick, ibid., 37:614, 1985;Ohno, 11 Mech. Aging Dev. 179, 1979; Ham and McKeehan, (1979) “Media andGrowth Requirements”, W. B. Jacoby and I. M. Pastan (eds), in: Methodsin Enzymology, Academic Press, N.Y., 58:44-93. The replicative life spanof cells is inversely proportional to the in vivo age of the donor(Martin et al., 23 Lab. Invest. 86, 1979; Goldstein et al., 64 Proc.Natl. Acad. Sci. USA 155, 1969; and Schneider and Mitsui, ibid.,73:3584, 1976), therefore cellular senescence is suggested to play animportant role in aging in vivo.

[0005] Cellular immortalization (the acquisition of unlimitedreplicative capacity) may be thought of as an abnormal escape fromcellular senescence, Shay et al., 196 Exp. Cell Res. 33, 1991. Normalhuman somatic cells appear to be mortal, i.e., have finite replicativepotential. In contrast, the germ line and malignant tumor cells areimmortal (have indefinite proliferative potential). Human cells culturedin vitro appear to require the aid of transforming viral oncoproteins tobecome immortal and even then the frequency of immortalization is 10⁻⁶to 10⁻⁷. Shay and Wright, 184 Exp. Cell Res. 109, 1989. A variety ofhypotheses have been advanced over the years to explain the causes ofcellular senescence. While examples of such hypotheses are providedbelow, there appears to be no consensus or universally acceptedhypothesis.

[0006] For example, the free radical theory of aging suggests that freeradical-mediated damage to DNA and other macromolecules is causative incritical loss of cell function (Harmon, 11 J. Gerontol. 298, 1956;Harmon, 16 J. Gerontol. 247, 1961). Harman says (Harman, 78 Proc. Natl.Acad. Sci. 7124, 1981) “aging is largely due to free radical reactiondamage . . . ”

[0007] Waste-product accumulation theories propose that the progressiveaccumulation of pigmented inclusion bodies (frequently referred to aslipofuscin) in aging cells gradually interferes with normal cellfunction (Strehler, 1 Adv. Geront. Res. 343, 1964; Bourne, 40 Prog.Brain Res. 187, 1973; Hayflick, 20 Exp. Gerontol. 145, 1985).

[0008] The gradual somatic mutation theories propose that theprogressive accumulation of genetic damage to somatic cells by radiationand other means impairs cell function and that without the geneticrecombination that occurs, for instance, during meiosis in the germ linecells, somatic cells lack the ability to proliferate indefinitely(Burnet, “Intrinsic Mutagenesis—A Genetic Approach to Aging”, Wile,N.Y., 1976; Hayflick, 27 Exp. Gerontol. 363, 1992). Theories concerninggenetically programmed senescence suggest that the expression ofsenescent-specific genes actively inhibit cell proliferation (Martin etal., 74 Am. J. Pathol. 137, 1974; Goldstein, 249 Science 1129, 1990).

[0009] Smith and Whitney, 207 Science 82, 1980, discuss a mechanism forcellular aging and state that their data is:

[0010] “compatible with the process of genetically controlled terminaldifferentiation. . . . The gradual decrease in proliferation potentialwould also be compatible with a continuous build up of damage or errors,a process that has been theorized. However, the wide variability indoubling potentials, especially in mitotic pairs, suggests an unequalledpartitioning of damage or errors at division.”

[0011] Shay et al., 27 Experimental Gerontology 477, 1992, and 196 Exp.Cell Res. 33, 1991 describe a two-stage model for human cell mortalityto explain the ability of Simian Virus 40 T-antigen to immortalize humancells. The mortality stage 1 mechanism (M1) is the target of certaintumor virus proteins, and an independent mortality stage 2 mechanism(M2) produces crisis and prevents these tumor viruses from directlyimmortalizing human cells. The authors utilized T-antigen driven by amouse mammary tumor virus promoter to cause reversible immortalizationof cells. The Simian Virus 40 T-antigen is said to extend thereplicative life span of human fibroblast by an additional 40-60%. Theauthors postulate that the M1 mechanism is overcome by T-antigen bindingto various cellular proteins, or inducing new activities to repress theM1 mortality mechanism. The M2 mechanism then causes cessation ofproliferation, even though the M1 mechanism is blocked. Immortality isachieved only when the M2 mortality mechanism is also disrupted.

[0012] It has also been proposed that the finite replicative capacity ofcells may reflect the work of a “clock” liked to DNA synthesis in thetelomere (end part) of the chromosomes. Olovnikov, 41 J. TheoreticalBiology 181, 1973, describes the theory of marginotomy to explain thelimitations of cell doubling potential in somatic cells. He states thatan:

[0013] “informative oligonucleotide, built into DNA after a telogene andcontrolling synthesis of a repressor of differentiation, might serve asa means of counting mitosis performed in the course of morphogenesis.Marginotomic elimination of such an oligonucleotide would present anappropriate signal for the beginning of further differentiation.Lengthening of the telogene would increase the number of possiblemitoses in differentiation.”

[0014] Harley et al., 345 Nature 458, 1990, state that the amount andlength of telomeric DNA in human fibroblasts decreases as a function ofserial passage during aging in vitro, and possibly in vivo, but do notknow whether this loss of DNA has a causal role in senescence. They alsostate:

[0015] “Tumour cells are also characterized by shortened telomeres andincreased frequency of aneuploidy, including telomeric associations. Ifloss of telomeric DNA ultimately causes cell-cycle arrest in normalcells, the final steps in this process may be blocked in immortalizedcells. Whereas normal cells with relatively long telomeres and asenescent phenotype may contain little or no telomerase activity, tumourcells with short telomeres may have significant telomerase activity.Telomerase may therefore be an effective target for anti-tumour drugs.

[0016] . . .

[0017] There are a number of possible mechanisms for loss of telomericDNA during ageing, including incomplete replication, degradation oftermini (specific or nonspecific), and unequal recombination coupled toselection of cells with shorter telomeres. Two features of our data arerelevant to this question. First, the decrease in mean telomere lengthis about 50 bp per mean population doubling and, second, thedistribution does not change substantially with growth state or cellarrest. These data are most easily explained by incomplete copying ofthe template strands at their 3′ termini. But the absence of detailedinformation about the mode of replication or degree of recombination attelomeres means that none of these mechanisms can be ruled out. Furtherresearch is required to determine the mechanism of telomere shorteningin human fibroblasts and its significance to cellular senescence.”[Citations]omitted.)

[0018] Hastie et al., 346 Nature 866, 1990, while discussing colon tumorcells, state that:

[0019] “[T]here is a reduction in the length of telomere repeat arraysrelative to the normal colonic mucosa from the same patient.

[0020] . . .

[0021] Firm figures are not available, but it is likely that the tissuesof a developed fetus result from 20-50 cell divisions, whereas severalhundred or thousands of divisions have produced the colonic mucosa andblood cells of 60-year old individuals. Thus the degree of telomerereduction is more or less proportional to the number of cell divisions.It has been shown that the ends of Drosophila chromosomes without normaltelomeres reduce in size by _(—)4 base pairs (bp) per cell division andthat the ends of yeast chromosomes reduce by a similar degree in amutant presumed to lack telomerase function. If we assume the same rateof reduction is occurring during somatic division in human tissues, thena reduction in TRA by 14 kb would mean that 3,500 ancestral celldivisions lead to the production of cells in the blood of a 60-year oldindividual; using estimates of sperm telomere length found elsewhere weobtain a value of 1,000-2,000. These values compare favourably withthose postulated for mouse blood cells. Thus, we propose that telomeraseis indeed lacking in somatic tissues. In this regard it is of interestto note that in maize, broken chromosomes are only healed in sporophytic(zygotic) tissues and not in endosperm (terminally differentiated),suggesting that telomerase activity is lacking in the differentiatedtissues.” [Citations omitted.]

[0022] The authors propose that in some tumors telomerase isreactivated, as proposed for HeLa cells in culture, which are known tocontain telomerase activity. But, they state:

[0023] “One alternative explanation for our observations is that intumours the cells with shorter telomeres have a growth advantage overthose with larger telomeres, a situation described for vegetative cellsof tetrahymena.” [Citations omitted.]

[0024] Harley, 256 Mutation Research 271, 1991, discusses observationsallegedly showing that telomeres of human somatic cells act as a mitoticclock shortening with age both in vitro and in vivo in a replicationdependent manner. He states:

[0025] “Telomerase activation may be a late, obligate event inimmortalization since many transformed cells and tumour tissues havecritically short telomeres. This, telomere length and telomeraseactivity appear to be markers of the replicative history andproliferative potential of cells; the intriguing possibility remainsthat telomere loss is a genetic time bomb and hence causally involved incell senescence and immortalization.

[0026] . . .

[0027] Despite apparently stable telomere length in various tumourtissues or transformed cell lines, this length was usually found to beshorter than those of the tissue of origin. These data suggest thattelomerase becomes activated as a late event in cell transformation, andthat cells could be viable (albeit genetically unstable) with shorttelomeres stably maintained by telomerase. If telomerase wasconstitutively present in a small fraction of normal cells, and thesewere the ones which survived crisis or became transformed, we wouldexpect to find a greater frequency of transformed cells with longtelomeres.”[Citations omitted.]

[0028] He proposes a hypothesis for human cell aging and transformationas “[a] semi-quantitative model in which telomeres and telomerase play acausal role in cell senescence and cancer” and proposes a model for thishypothesis.

[0029] De Langa et al., 10 Molecular and Cellular Biology 518, 1990,generally discuss the structure of human chromosome ends or telomeres.They state:

[0030] “we do not know whether telomere reduction is strictly coupled tocellular proliferation. If the diminution results from incompletereplication of the telomere, such a coupling would be expected; however,other mechanisms, such as exonucleolytic degradation, may operateindependent of cell division. In any event, it is clear that themaintenance of telomeres is impaired in somatic cells. An obviouscandidate activity that may be reduced or lacking is telomerase. A humantelomerase activity that can add TTAGGG repeats to G-rich primers hasrecently been identified (G. Morin, personal communication).Interestingly, the activity was demonstrated in extracts of HeLa cells,which we found to have exceptionally long telomeres. Other cell typeshave not been tested yet, but such experiments could now establishwhether telomerase activity is (in part) responsible for the dynamics ofhuman chromosome ends.”

[0031] Starling et al., 18 Nucleic Acids Research 6881, 1990, indicatethat mice have large telomeres and discusses this length in relationshipto human telomeres. They state:

[0032] “Recently it has been shown that there is reduction in TRA lengthwith passage number of human fibroblasts in vitro and that cells in asenescent population may lack telomeres at some ends altogether. Thus invitro, telomere loss may play a role in senescence, a scenario for whichthere is evidence in S. cerevisae and Tetrahymena.

[0033] Some of the mice we have been studying are old in mouse terms,one and a half years, yet they still have TRA's greater than 30 kb inall tissues studied. In humans, telomeres shorten with age at a rate of100 bp per year, hence, it is conceivable that the same is happening inthe mouse, but the removal of a few 100 bps of terminal DNA during itslifetime would not be detectable.” [Citations omitted.]

[0034] D'Mello and Jazwinski, 173 J. Bacteriology 6709, 1991, state:

[0035] “We propose that during the life span of an organism, telomereshortening does not play a role in the normal aging process. However,mutations or epigenetic changes that affect the activity of thetelomerase, like any other genetic change, might affect the life span ofthe individual in which they occur.

[0036] . . .

[0037] In summary, the telomere shortening with age observed in humandiploid fibroblasts may not be a universal phenomenon. Further studiesare required to examine telomere length and telomerase activity not onlyin different cell types as they age but also in the same cell type indifferent organisms with differing life spans. This would indicatewhether telomere shortening plays a causal role in the senescence of aparticular cell type or organism.”

[0038] Hiyama et al., 83 Jpn. J. Cancer Res. 159, 1992, provide findingsthat “suggest that the reduction of telomeric repeats is related to theproliferative activity of neuroblastoma cells and seems to be a usefulindicator of the aggressiveness of neuroblastoma. . . . Although we donot know the mechanism of the reduction and the elongation of telomericrepeats in neuroblastoma, we can at least say that the length oftelomeric repeats may be related to the progression and/or regression ofneuroblastoma.”

[0039] Counter et al., 11 EMBO J. 1921, 1992, state “loss of telomericDNA during cell proliferation may play a role in ageing and cancer.”They propose that the expression of telomerase is one of the eventsrequired for a cell to acquire immortality and note that:

[0040] This model may have direct relevance to tumourigenesis in vivo.For example, the finite lifespan of partially transformed (pre-immortal)cells which lack telomerase might explain the frequent regression oftumours after limited growth in vivo. In bypassing the checkpointrepresenting normal replicative senescence, transformation may confer anadditional 20-40 population doubling during which an additional ≈2 kbpof telomeric DNA is lost. Since 20-40 doubling (10⁶-10¹² cells in aclonal population) potentially represents a wide range of tumour sizes,it is possible that many benign tumours may lack telomerase andnaturally regress when telomeres become critically shortened. We predictthat more aggressive, perhaps metastatic tumours would contain immortalcells which express telomerase. To test this hypothesis, we arecurrently attempting to detect telomerase in a variety of tumour tissuesand to correlate activity with proliferative potential. Anti-telomerasedrugs or mechanisms to repress telomerase expression could be effectiveagents against tumours which depend upon the enzyme for maintenance oftelomeres and continued cell growth.

[0041] Levy et al., 225 J. Mol. Biol. 951, 1992, state that:

[0042] “Although it has not been proven that telomere loss contributesto senescence of multicellular organisms, several lines of evidencesuggest a causal relationship may exist.

[0043] . . .

[0044] It is also possible that telomere loss with age is significant inhumans, but not in mice.” [Citations omitted.]

[0045] Windle and McGuire, 33 Proceedings of the American Associationfor Cancer Research 594, 1992, discuss the role of telomeres and statethat:

[0046] “These and other telomere studies point in a new directionregarding therapeutic targets and strategies to combat cancer. If thecell can heal broken chromosomes preventing genomic disaster, then theremay be a way to facilitate or artificially create this process. Thiscould even provide a preventive means of stopping cancer which could beparticularly applicable in high risk patients. The difference intelomere length in normal versus tumor cells also suggests a strategywhere the loss of telomeres is accelerated. Those cells with theshortest telomeres, such as those of tumor metastasis would be the mostsusceptible.”

[0047] Goldstein, 249 Science 1129, 1990, discusses various theories ofcellular senescence including that of attrition of telomeres. He states:

[0048] “However, such a mechanism is not easily reconciled with thedominance of senescent HDF over young HDF in fusion hybrids,particularly in short-term heterokaryons. One could again invoke theconcept of dependence and the RAD9 gene example, such that complete lossof one or a few telomeres leads to the elaboration of a negative signalthat prevents initiation of DNA synthesis, thereby mimicking thedifferentiated state. This idea, although speculative, would not onlyexplain senescent replicative arrest but also the chromosomalaberrations observed in senescent HDS that would specifically ensueafter loss of telomeres.” [Citations omitted.]

[0049] The role of telomere loss in cancer is further discussed byJankovic et al. and Hastie et al., both at 350 Nature 1991, in whichJankovic indicates that telomere shortening is unlikely to significantlyinfluence carcinogenesis in men and mice. Hastie et al. agree that iftelomere reduction does indeed reflect cell turnover, this phenomenon isunlikely to play a role in pediatric tumors, and those of the centralnervous system. Hastie et al., however, feel “our most original andinteresting conclusion was that telomere loss may reflect the number ofcell division in a tissue history, constituting a type of clock.”

[0050] Kipling and Cooke, 1 Human Molecular Genetics 3, 1992, state:

[0051] “It has been known for some years that telomeres in humangermline cells (e.g. sperm) are longer than those in somatic tissue suchas blood. One proposed explanation for this is the absence of telomererepeat addition (i.e. absence of telomerase activity) in somatic cells.If so, incomplete end replication would be expected to result in theprogressive loss of terminal repeats as somatic cells undergo successiverounds of division. This is indeed what appears to happen in vivo forhumans, with both blood and skin cells showing shorter telomeres withincreasing donor age, and telomere loss may contribute to the chromosomeaberrations typically seen in senescent cells. Senescence and themeasurement of cellular time is an intriguingly complex subject and itwill be interesting to see to what extent telomere shortening has acausal role. The large telomeres possessed by both young and old micewould seem to preclude a simple relationship between telomere loss andageing, but more elaborate schemes cannot be ruled out.”[Citationsomitted.]

[0052] Greider, 12 BioEssays 363, 1990, provides a review of therelationship between telomeres, telomerase, and senescence. Sheindicates that telomerase contains an RNA component which provides atemplate for telomere repeat synthesis. She notes that anoligonucleotide “which is complementary to the RNA up to and includingthe CAACCCCAA sequence, competes with d(TTGGGG)n primers and inhibitstelomerase in vitro” (citing Greider and Blackburn, 337 Nature 331,1989). She also describes experiments which she believes “provide directevidence that telomerase is involved in telomere synthesis in vivo.” Shegoes on to state:

[0053] “Telomeric restriction fragments in many transformed cell linesare much shorter than those in somatic cells. In addition, telomerelength in tumor tissues is significantly shorter than in the adjacentnon-tumor tissue. When transformed cell lines are passaged in vitrothere is no change in telomere length. Thus if untransformed cells lackthe ability to maintain a telomere length equilibrium, most transformedcells appear to regain it and to reset the equilibrium telomere lengthto a size shorter than seen in most tissues in vivo. The simplestinterpretation of these data is that enzymes, such as telomerase,involved in maintaining telomere length may be required for growth oftransformed cells and not required for normal somatic cell viability.This suggests that telomerase may be a good target for anti-tumordrugs.” [Citations omitted.]

[0054] Blackburn, 350 Nature 569, 1991, discusses the potential for drugaction at telomeres stating:

[0055] “The G-rich strand of the telomere is the only essentialchromosomal DNA sequence known to be synthesized by the copying of aseparate RNA sequence. This unique mode of synthesis, and the specialstructure and behavior of telomeric DNA, suggest that telomere synthesiscould be a target for selective drug action. Because telomerase activityseems to be essential for protozoans or yeast, but not apparently formammalian somatic cells, I propose that telomerase should be explored asa target for drugs against eukaryotic pathogenic or parasiticmicroorganisms, such as parasitic protozoans or pathogenic yeasts. Adrug that binds telomerase selectively, either through itsreverse-transcriptase or DNA substrate-binding properties, shouldselectively act against prolonged maintenance of the dividing lowereukaryote, but not impair the mammalian host over the short term,because telomerase activity in its somatic cells may normally be low orabsent. Obvious classes of drugs to investigate are those directedspecifically against reverse transcriptases as opposed to other DNA orRNA polymerases, and drugs that would bind telomeric DNA itself. Thesecould include drugs that selectively bind the G°G base-paired forms ofthe G-rich strand protrusions at the chromosome termini, or agents whichstabilize an inappropriate G°G base-paired form, preventing it fromadopting a structure necessary for proper function in vivo. Telomereshave been described as the Achilles heel of chromosomes: perhaps it isthere that drug strategies should now be aimed.” [Citations omitted.]

[0056] Lundblad and Blackburn, 73 Cell 347, 1993, discuss alternativepathways for maintainance of yeast telomers, and state that:

[0057] “. . . the work presented in this paper demonstrates that adefect in telomere replication need not result in the death of all cellsin a population, suggesting that telomere loss and its relationship tomammalian cellular senescence may have to be examined further.”

[0058] Other review articles concerning telomeres include Blackburn andSzostak, 53 Ann. Rev. Biochem. 163, 1984; Blackburn, 350 Nature 569,1991; Greider, 67 Cell 645, 1991, and Moyzis 265 Scientific American 48,1991. Relevant articles on various aspects of telomeres include Cookeand Smith, Cold Spring Harbor Symposia on Quantitative Biology Vol. LI,pp. 213-219; Morin, 59 Cell 521, 1989; Blackburn et al., 31 Genome 553,1989; Szostak, 337 Nature 303, 1989; Gall, 344 Nature 108, 1990;Henderson et al., 29 Biochemistry 732, 1990; Gottschling et al., 63 Cell751, 1990; Harrington and Grieder, 353 Nature 451, 1991; Muller et al.,67 Cell 815, 1991; Yu and Blackburn, 67 Cell 823, 1991; and Gray et al.,67 Cell 807, 1991. Other articles or discussions of some relevanceinclude Lundblad and Szostak, 57 Cell 633, 1989; and Yu et al., 344Nature 126, 1990.

SUMMARY OF THE INVENTION

[0059] This invention concerns methods for therapy and diagnosis ofcellular senescence and immortalization utilizing techniques associatedwith control of telomere length and telomerase activity. Therapeuticstrategies of this invention include reducing the rate or absoluteamount of telomere repeat length loss or increasing the telomere repeatlength during cell proliferation, thereby providing for the postponementof cellular senescence and reducing the level of chromosomal fusions andother chromosomal aberrations. In addition, inhibition of telomeraseactivity in vivo or in vitro may be used to control diseases associatedwith cell immortality, such as neoplasia, and pathogenic parasites.

[0060] Applicant has determined that the inhibition of telomereshortening in a cell in vitro is causally related to increasing thelength of the replicative lifespan of that cell. Applicant has alsodetermined that inhibition of telomerase activity in a cell in vitro iscausally related to reducing the ability of that cell to proliferate inan immortal manner. Thus, applicant is the first to provide data whichclearly indicates that inhibition of telomere shortening in vivo or invitro, and that inhibition of telomerase activity in vivo or in vitro,is therapeutically beneficial. Prior to applicant's experiments, asindicated above, there was no consensus by those in the art that onecould predict that such experiments would provide the data observed byapplicant, or that such manipulations would have therapeutic utility.

[0061] The invention also concerns the determination of cellular statusby diagnostic techniques that analyze telomere length and telomeraseactivity, as a diagnostic of cellular capacity for proliferation. Assaysfor telomere length are performed to provide useful information on therelative age and remaining proliferative capability of a wide variety ofcell types in numerous tissues. Sequences are also described from thetelomeres of budding yeasts which are highly variable from strain tostrain and provide sequences for oligonucleotide probes that wouldenable the rapid identification of yeast strains, and in the case ofhuman and veterinary pathogens, the diagnosis of the strain of thepathogen.

[0062] Telomerase activity and the presence of the enzyme is used as amarker for diagnosing and staging neoplasia and detecting pathogenicparasites. Applicant's experiments have, for the first time, determineda correlation between telomerase activity and the tumor cell phenotype,as well as a correlation between telomere length and the in vivo agedstatus of cells. As noted above, there was no consensus in the art thatone could predict that such a relationship existed. In contrast,applicant has defined this relationship, and thus has now defined usefuldiagnostic tools by which to determine useful clinical data. Such datacan be used to define a therapeutic protocol, or the futility of such aprotocol.

[0063] Thus, in a first aspect, the invention features methods for thetreatment of a condition associated with cellular senescence orincreased rate of proliferation of a cell (e.g., telomere repeat lossassociated with cell proliferation in the absence of telomerase). Afirst method involves administering to the cell a therapeuticallyeffective amount of an agent active to reduce loss of telomeric repeatsduring its proliferation. Such therapeutics may be especially applicableto conditions of increased rate of cell proliferation.

[0064] By “increased rate of proliferation” of a cell is meant that thecell has a higher rate of cell division compared to normal cells of thatcell type, or compared to normal cells within other individuals of thatcell type. Examples of such cells include the CD4⁺ cells of HIV-infectedindividuals (see example below), connective tissue fibroblastsassociated with degenerative joint diseases, age-related maculardegeneration, astrocytes associated with Alzheimer's Disease andendothelial cells associated with atherosclerosis (see example below).In each case, one particular type of cell or a group of cells is foundto be replicating at an increased level compared to surrounding cells inthose tissues, or compared to normal individuals, e.g., individuals notinfected with the HIV virus. Thus, the invention features administeringto those cells an agent which reduces loss of telomere length in thosecells while they proliferate. The agent itself need not slow theproliferation process, but rather allow that proliferation process tocontinue for more cell divisions than would be observed in the absenceof the agent. The agent may also be useful to slow telomere repeat lossoccurring during normal aging (wherein the cells are proliferating at anormal rate and undergoing senescence late in life), and for reducingtelomere repeat loss while expanding cell number ex vivo for cell-basedtherapies, e.g., bone marrow transplantation following gene therapy.

[0065] As described herein, useful agents can be readily identified bythose of ordinary skill in the art using routine screening procedures.For example, a particular cell having a known telomere length is chosenand allowed to proliferate, and the length of telomere is measuredduring proliferation. Agents which are shown to reduce the loss oftelomere length during such proliferation are useful in this invention.Particular examples of such agents are provided below. For example,oligonucleotides which are able to promote synthesis of DNA at thetelomere ends are useful in this invention. In addition, telomerase maybe added to a cell either by gene therapy techniques, or by of theenzyme or its equivalent into a cell administration, e.g., by injectionor lipojection.

[0066] A second method for the treatment of cellular senescence involvesthe use of an agent to derepress telomerase in cells where the enzyme isnormally repressed. Telomerase activity is not detectable in any normalhuman somatic cells, but is detectable in cells that have abnormallyreactivated the enzyme during the transformation of a normal cell intoan immortal tumor cell. Telomerase activity may therefore be appropriateonly in germ line cells and some stem cell populations (though there iscurrently no evidence of the latter in human tissues). Since the loss oftelomeric repeats leading to senescence in somatic cells is occuring dueto the absence of adequate telomerase activity, agents that have theeffect of activating telomerase would have the effect of adding arraysof telomeric repeats to telomeres, thereby imparting to mortal somaticcells increased replicative capacity, and imparting to senescent cellsthe ability to proliferate and appropriately exit the cell cycle (in theabsence of growth factor stimulation with associated appropriateregulation of cell cycle-linked genes typically inappropriatelyexpressed in senescence e.g., collagenase, urokinase, and other secretedproteases and protease inhibitors). Such factors to derepress telomerasemay be administered transiently or chronically to increase telomerelength, and then removed, thereby allowing the somatic cells to againrepress the expression of the enzyme utilizing the natural mechanisms ofrepression.

[0067] Such activators of telomerase may be found by screeningtechniques utilizing human cells that have the M1 mechanism ofsenescence abrogated by means of the expression of SV40 T-antigen. Suchcells when grown to crisis, wherein the M2 mechanism is preventing theirgrowth, will proliferate in response to agents that derepresstelomerase. Such activity can be scored as the incorporation ofradiolabeled nucleotides or proliferating clones can be selected for ina colony forming assay.

[0068] Such activators of telomerase would be useful as therapeuticagents to forestall and reverse cellular senescence, including but notlimited to conditions associated with cellular senescence, e.g., (a)cells with replicative capacity in the central nervous system, includingastrocytes, endothelial cells, and fibroblasts which play a role in suchage-related diseases as Alzheimer's disease, Parkinson's disease,Huntington's disease, and stroke, (b) cells with finite replicativecapacity in the integument, including fibroblasts, sebaceous glandcells, melanocytes, keratinocytes, Langerhan's cells, and hair folliclecells which may play a role in age-related diseases of the integumentsuch as dermal atrophy, elastolysis and skin wrinkling, sebaceous glandhyperplasia, senile lentigo, graying of hair and hair loss, chronic skinulcers, and age-related impairment of wound healing, (c) cells withfinite replicative capacity in the articular cartilage, such aschondrocytes and lacunal and synovial fibroblasts which play a role indegenerative joint disease, (d) cells with finite replicative capacityin the bone, such as osteoblasts and osteoprogenitor cells which play arole in osteoporosis, (e) cells with finite replicative capacity in theimmune system such as B and T lymphocytes, monocytes, neutrophils,eosinophils, basophils, NK cells and their respective progenitors, whichmay play a role in age-related immune system impairment, (f) cells witha finite replicative capacity in the vascular system includingendothelial cells, smooth muscle cells, and adventitial fibroblastswhich may play a role in age-related diseases of the vascular systemincluding atherosclerosis, calcification, thrombosis, and aneurysms, and(g) cells with a finite replicative capacity in the eye such aspigmented epithelium and vascular endothelial cells which may play animportant role in age-related macular degeneration.

[0069] In a second aspect, the invention features a method for treatmentof a condition associated with an elevated level of telomerase activitywithin a cell. The method involves administering to that cell atherapeutically effective amount of an inhibitor of telomerase activity.

[0070] The level of telomerase activity can be measured as describedbelow, or by any other existing methods or equivalent methods. By“elevated level” of such activity is meant that the absolute level oftelomerase activity in the particular cell is elevated compared tonormal cells in that individual, or compared to normal cells in otherindividuals not suffering from the condition. Examples of suchconditions include cancerous conditions, or conditions associated withthe presence of cells which are not normally present in that individual,such as protozoan parasites or opportunistic pathogens, which requiretelomerase activity for their continued replication. Administration ofan inhibitor can be achieved by any desired means well known to those ofordinary skill in the art.

[0071] In addition, the term “therapeutically effective amount” of aninhibitor is a well recognized phrase. The amount actually applied willbe dependent upon the individual or animal to which treatment is to beapplied, and will preferably be an optimized amount such that aninhibitory effect is achieved without significant side-effects (to theextent that those can be avoided by use of the inhibitor). That is, ifeffective inhibition can be achieved with no side-effects with theinhibitor at a certain concentration, that concentration should be usedas opposed to a higher concentration at which side-effects may becomeevident. If side-effects are unavoidable, however, the minimum amount ofinhibitor that is necessary to achieve the inhibition desired may haveto be used.

[0072] By “inhibitor” is simply meant any reagent, drug or chemicalwhich is able to inhibit a telomerase activity in vitro or in vivo. Suchinhibitors can be readily identified using standard screening protocolsin which a cellular extract or other preparation having telomeraseactivity is placed in contact with a potential inhibitor, and the levelof telomerase activity measured in the presence or absence of theinhibitor, or in the presence of varying amounts of inhibitor. In thisway, not only can useful inhibitors be identified, but the optimum levelof such an inhibitor can be determined in vitro for further testing invivo.

[0073] One example of a suitable telomerase inhibitor assay is carriedout in 96-well microtiter plates. One microtiter plate is used to makedilutions of the test compounds, while another plate is used for theactual assay. Duplicate reactions of each sample are performed. Amixture is made containing the appropriate amount of buffer, templateoligonucleotide, and Tetrahymena or human telomerase extract for thenumber of the samples to be tested, and aliquots are placed in the assayplate. The test compounds are added individually and the plates arepre-incubated at 30° C. ³²P-dGTP is then added and the reaction allowedto proceed for 10 minutes at 30° C. The total volume of each reaction is10 μl. The reaction is then terminated by addition of Tris and EDTA, andhalf the volume (5 μl) spotted onto DE81 filter paper. The samples areallowed to air dry, and the filter paper is rinsed in 0.5 M NaPhosphateseveral times to wash away the unincorporated labeled nucleotide. Afterdrying, the filter paper is exposed to a phosphor imaging plate and theamount of signal quantitated. By comparing the amount of signal for eachof the test samples to control samples, the percent of inhibition can bedetermined.

[0074] In addition, a large number of potentially useful inhibitors canbe screened in a single test, since it is inhibition of telomeraseactivity that is desired. Thus, if a panel of 1,000 inhibitors is to bescreened, all 1,000 inhibitors can potentially be placed into microtiterwells. If such an inhibitor is discovered, then the pool of 1,000 can besubdivided into 10 pools of 100 and the process repeated until anindividual inhibitor is identified. As discussed herein, oneparticularly useful set of inhibitors includes oligonucleotides whichare able to either bind with the RNA present in telomerase or able toprevent binding of that RNA to its DNA target or one of the telomeraseprotein components. Even more preferred are those oligonucleotides whichcause inactivation or cleavage of the RNA present in a telomerase. Thatis, the oligonucleotide is chemically modified or has enzyme activitywhich causes such cleavage. The above screening may include screening ofa pool of many different such oligonucleotide sequences.

[0075] In addition, a large number of potentially useful compounds canbe screened in extracts from natural products. Sources of such extractscan be from a large number of species of fungi, actinomyces, algae,protozoa, plants, and bacteria. Those extracts showing inhibitoryactivity can then be analyzed to isolate the active molecule.

[0076] In related aspects, the invention features pharmaceuticalcompositions which include therapeutically effective amounts of theinhibitors or agents described above, in pharmaceutically acceptablebuffers much as described below. These pharmaceutical compositions mayinclude one or more of these inhibitors or agents, and beco-administered with other drugs. For example, AZT is commonly used fortreatment of HIV, and may be co-administered with an inhibitor or agentof the present invention.

[0077] In a related aspect, the invention features a method forextending the ability of a cell to replicate. In this method, areplication extending amount of an agent which is active to reduce lossof telomere length within the cell is provided during cell replication.As will be evident to those of ordinary skill in the art, this agent issimilar to that useful for treatment of a condition associated with anincreased rate of proliferation of a cell. However, this method isuseful for the treatment of individuals not suffering from anyparticular condition, but in which one or more cell types are limitingin that patient, and whose life can be extended by extending the abilityof those cells to continue replication. That is, the agent is added todelay the onset of cell senescence characterized by the inability ofthat cell to replicate further in an individual. One example of such agroup of cells includes lymphocytes present in patients suffering fromDowns Syndrome (although treatment of such cells may also be useful inindividuals not identified as suffering from any particular condition ordisease, but simply recognizing that one or more cells, or collectionsof cells are becoming limiting in the life span of that individual).

[0078] It is notable that administration of such inhibitors or agents isnot expected to be detrimental to any particular individual. However,should gene therapy be used to introduce a telomerase into anyparticular cell population, or other means be used to reversiblyde-repress telomerase activity in somatic cells, care should be taken toensure that the activity of that telomerase is carefully regulated, forexample, by use of a promoter which can be regulated by the nutrition ofthe patient. Thus, for example, the promoter may only be activated whenthe patient eats a particular nutrient, and is otherwise inactive. Inthis way, should the cell population become malignant, that individualmay readily inactivate telomerase of the cell and cause it to becomemortal simply by no longer eating that nutrient.

[0079] In a further aspect, the invention features a method fordiagnosis of a condition in a patient associated with an elevated levelof telomerase activity within a cell. The method involves determiningthe presence or amount of telomerase within the cells in that patient.

[0080] In yet another aspect, the invention features a method fordiagnosis of a condition associated with an increased rate ofproliferation in that cell in an individual. Specifically, the methodinvolves determining the length of telomeres within the cell. Thevarious conditions for which diagnosis is possible are described above.As will be exemplified below, many methods exist for measuring thepresence or amount of telomerase within a cell in a patient, and fordetermining the length of telomeres within the cell. It will be evidentthat the presence or amount of telomerase may be determined within anindividual cell, and for any particular telomerase activity (whether itbe caused by one particular enzyme or a plurality of enzymes). Those inthe art can readily formulate antibodies or their equivalent todistinguish between each type of telomerase present within a cell, orwithin an individual. In addition, the length of telomeres can bedetermined as an average length, or as a range of lengths much asdescribed below. Each of these measurements will give preciseinformation regarding the status of any particular individual.

[0081] Thus, applicant's invention has two prongs—a diagnostic and atherapeutic prong. These will now be discussed in detail.

[0082] The therapeutic prong of the invention is related to the nowclear observation that the ability of a cell to remain immortal lies inthe ability of that cell to maintain or increase the telomere length ofchromosomes within that cell. Such a telomere length can be maintainedby the presence of sufficient activity of telomerase, or an equivalentenzyme, within the cell. Thus, therapeutic approaches to reducing thepotential of a cell to remain immortal focus on the inhibition oftelomerase activity within those cells in which it is desirable to causecell death. Examples of such cells include cancerous cells, which areone example of somatic cells which have regained the ability to expresstelomerase, and have become immortal. Applicant has now shown that suchcells can be made mortal once more by inhibition of telomerase activity.As such, inhibition can be achieved in a multitude of ways including, asillustrated below, the use of oligonucleotides which, in some manner,block the ability of telomerase to extend telomeres in vivo.

[0083] Thus, oligonucleotides can be designed either to bind to atelomere (to block the ability of telomerase to bind to that telomere,and thereby extend that telomere), or to bind to the residentoligonucleotide (RNA) present in telomerase to thereby block telomeraseactivity on any nucleic acid (telomere). Such oligonucleotides may beformed from naturally occurring nucleotides, or may include modifiednucleotides to either increase the stability of the therapeutic agent,or cause permanent inactivation of the telomerase, e.g., the positioningof a chain terminating nucleotide at the 3′ end of the molecule of anucleotide with a reactive group capable of forming a covalent bond withtelomerase. Such molecules may also include ribozyme sequences. Inaddition, non-oligonucleotide based therapies can be readily devised byscreening for those molecules which have an ability to inhibittelomerase activity in vitro, and then using those molecules in vivo.Such a screen is readily performed and will provide a large number ofuseful therapeutic molecules. These molecules may be used for treatmentof cancers, of any type, including solid tumors and leukemias (includingthose in which cells are immortalized, including: apudoma, choristoma,branchioma, malignant carcinoid syndrome, carcinoid heart disease,carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal,Ehrlich tumor, in situ, Krebs 2, merkel cell, mucinous, non-small celllung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic,squamous cell, and transitional cell), histiocytic disorders, leukemia(e.g., b-cell, mixed-cell, null-cell, T-cell, T-cell chronic,HTLV-II-associated, lyphocytic acute, lymphocytic chronic, mast-cell,and myeloid), histiocytosis malignant, Hodgkin's disease,immunoproliferative small, non-Hodgkin's lymphoma, plasmacytoma,reticuloendotheliosis, melanoma, chondroblastoma, chondroma,chondrosarcoma, fibroma, fibrosarcoma, giant cell tumors, histiocytoma,lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma,osteosarcoma, Ewing's sarcoma, synovioma, adenofibroma, adenolymphoma,carcinosarcoma, chordoma, craniopharyngioma, dysgerminoma, hamartoma,mesenchymoma, mesonephroma, myosarcoma, ameloblastoma, cementoma,odontoma, teratoma, thymoma, trophoblastic tumor, adenocarcinoma,adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma,cystadenoma, granulosa cell tumor, gynandroblastoma, hepatoma,hidradenoma, islet cell tumor, leydig cell tumor, papilloma, sertolicell tumor, theca cell tumor, leiomyoma, leiomvosarcoma, myoblastoma,myoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma, ependymoma,ganglioneuroma, glioma, medulloblastoma, meningioma, neurilemmoma,neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma,paraganglioma nonchromaffin, angiokeratoma, angiolymphoid hyperplasiawith eosinophilia, angioma sclerosing, angiomatosis, glomangioma,hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma,lymphangioma, lymphangiomyoma, lymphangiosarcoma, pinealoma,carcinosarcoma, chondrosarcoma, cystosarcoma phyllodes, fibrosarcoma,hemangiosarcoma, leiomyosarcoma, leukosarcoos arliposarcoma,lymphangiosarcoma, myosarcoma, myxosarcoma, ovarian carcinoma,rhabdomyosarcoma, sarcoma (e.g., Ewing's, experimental, Kaposi's, andmast-cell), neoplasms (e.g., bone, breast, digestive system, colorectal,liver, pancreatic, pituitary, testicular, orbital, head and neck,central nervous system, acoustic, pelvic, respiratory tract, andurogenital), neurofibromatosis, and cervical dysplasia), and fortreatment of other conditions in which cells have become immortalized.

[0084] Applicant has also determined that it is important to slow theloss of telomere sequences, in particular, cells in association withcertain diseases (although such treatment is not limited to this, andcan be used in normal aging and ex vivo treatments). For example, somediseases are manifest by the abnormally fast rate of proliferation ofone or more particular groups of cells. Applicant has determined that itis the senescence of those groups of cells at an abnormally early age(compared to the age of the patient), that eventually leads to death ofthat patient. One example of such a disease is AIDS, in which death iscaused by the early senescence of CD4⁺ cells. It is important to notethat such cells age, not because of abnormal loss of telomere sequences(although this may be a factor), but rather because the replicative rateof the CD4⁺ cells is increased such that telomere attrition is caused ata greater rate than normal for that group of cells. Thus, applicantprovides therapeutic agents which can be used for treatment of suchdiseases, and also provides a related diagnostic procedure by whichsimilar diseases can be detected so that appropriate therapeuticprotocols can be devised and followed.

[0085] Specifically, the loss of telomeres within any particular cellpopulation can be reduced by provision of an oligonucleotide whichreduces the extent of telomere attrition during cell division, and thusincreases the number of cell divisions that may occur before a cellbecomes senescent. Other reagents, for example, telomerase, may beprovided within a cell in order to reduce telomere loss, or to make thatcell immortal. Those of ordinary skill in the art will recognize thatother enzymatic activities may be used to enhance the lengthening oftelomeres within such cells, for example, by providing certain viralsequences which activate telomerase or can otherwise function tosynthesize telomere sequences within a cell. In addition, equivalentsuch molecules, or other molecules may be readily screened to determinethose that will reduce loss of telomeres. Such screens may occur invitro, and the therapeutic agents discovered by such screening utilizedin the above method in vivo.

[0086] Other therapeutic treatments relate to the finding of unusualtelomeric DNA sequences in a group of fungi, specifically a group ofbudding yeasts that includes some pathogens—Candida albicans, Candidatropicalis and Candida paratropicalis—as well as nonpathogenic fungi.These results are described in more detail below. Drugs or chemicalagents can be used to specifically exploit the unusual nature of thetelomeric DNA of fungi. This includes the introduction of antisensepolynucleotides specific to the telomeric repeat DNA sequences, in orderto block telomere synthesis in these and any related pathogens. Such ablock will lead to fungal death.

[0087] This approach is advantageous because of the unusual nature ofthe telomeric DNA in these fungi. The unusually high DNA sequencecomplexity of the telomeric repeats of these fungi provides specificity,and potential for minimal side effects, of the antifungal agent or theantisense DNA or RNA.

[0088] Agents that are potentially useful antifungal agents include:AZT, d4T, ddI, ddC, and ddA. The telomere synthesis of these fungi isexpected to show differential inhibition to these drugs, and in somecases to be more sensitive than the telomere synthesis in the human orother animal or plant host cells.

[0089] We performed a preliminary test of the use of antisensetechniques in living fungal cells. A stretch of 40 bp of telomeric DNAsequence, imbedded in a conserved sequence flanking a region of Candidaalbicans chromosomal DNA, was introduced on a circular molecule intoCandida albicans cells. The transformed cells had high copy numbers ofthe introduced telomeric DNA sequence. 10% of the transformantsexhibited greatly (˜3-fold) increased length of telomeric DNA. Thisresult indicates that telomeric DNA can be modulated in vivo byintroduction of telomeric sequence polynucleotides into cells. Thisdemonstrates the need to test a particular oligonucleotide to ensurethat it has the desired activity.

[0090] With regard to diagnostic procedures, examples of such proceduresbecome evident from the discussion above with regard to therapy.Applicant has determined that the length of the telomere is indicativeof the life expectancy of a cell containing that telomere, and of anindividual containing that cell. Thus, the length of a telomere isdirectly correlated to the life span of an individual cell. As discussedabove, certain populations of cells may lose telomeres at a greater ratethan the other cells within an individual, and those cells may thusbecome age-limiting within an individual organism. However, diagnosticprocedures can now be developed (as described herein) which can be usedto indicate the potential life span of any individual cell type, and tofollow telomere loss so that a revised estimate to that life span can bemade with time.

[0091] In certain diseases, for example, the AIDS disease discussedabove, it would, of course, be important to follow the telomere lengthin CD4⁺ cells. In addition, the recognition that CD4⁺ cells are limitingin such individuals allows a therapeutic protocol to be devised in whichCD4⁺ cells can be removed from the individual at an early age when AIDSis first detected, stored in a bank, and then reintroduced into theindividual at a later age when that individual no longer has therequired CD4⁺ cells available. These cells can be expanded in number inthe presence of agents which slow telomere repeat loss, e.g., C-richtelomeric oligonucleotides or agents to transiently de-represstelomerase to ensure that cells re-administered to the individual havemaximum replicative capacity. Thus, an individual's life can be extendedby a protocol involving continued administration of that individual'slimiting cells at appropriate time points. These appropriate points canbe determined by following CD4⁺ cell senescence, or by determining thelength of telomeres within such CD4⁺ cells (as an indication of whenthose cells will become senescent). In the case of AIDS, there may bewaves of senescent telomere length in peripheral blood lymphocytes withbone marrow tem cells still having replicative capacity. In this way,rather than wait until a cell becomes senescent (and thereby putting anindividual at risk of death) telomere length may be followed until thelength is reduced below that determined to be pre-senescent, and therebythe timing of administration of new CD4⁺ cells or colony stimulatingfactors can be optimized.

[0092] Thus, the diagnostic procedures of this invention includeprocedures in which telomere length in different cell populations ismeasured to determine whether any particular cell population is limitingin the life span of an individual, and then determining a therapeuticprotocol to insure that such cells are no longer limiting to thatindividual. In addition, such cell populations may be specificallytargeted by specific drug administration to insure that telomere lengthloss is reduced, as discussed above.

[0093] Other diagnostic procedures include measurement of telomeraseactivity as an indication of the presence of immortal cells within anindividual. A more precise measurement of such immortality is thepresence of the telomerase enzyme itself. Such an enzyme can be readilydetected using standard procedures, including assay of telomeraseactivities, but also by use of antibodies to telomerase, or by use ofoligonucleotides that hybridize to the nucleic acid (template RNA)present in telomerase, or DNA or RNA probes for the mRNAs of telomeraseproteins. Immunohistochemical and insitu hybridization techniques allowthe precise identification of telomerase positive cells in histologicalspecimens for diagnostic and prognostic tests. The presence oftelomerase is indicative of cells which are immortal and frequentlymetastatic, and such a diagnostic allows pinpointing of such metastaticcells, much as CD44 is alleged to do so. See, Leff, 3(217) BioWorldToday 1, 3, 1992.

[0094] It is evident that the diagnostic procedures of the presentinvention provide the first real method for determining how far certainindividuals have progressed in a certain disease. For example, in theAIDS disease, this is the first methodology which allows priordetermination of the time at which an HIV positive individual willbecome immunocompromised. This information is useful for determining thetiming of drug administration, such as AZT administration, and will aidin development of new drug regimens or therapies. In addition, thedetermination of the optimum timing of administration of certain drugswill reduce the cost of treating an individual, reduce the opportunityfor the drug becoming toxic to the individual, and reduce the potentialfor the individual developing resistance to such a drug.

[0095] In other related aspects, the invention features a method fortreatment of a disease or condition associated with cell senescence, byadministering a therapeutically effective amount of an agent active toderepress telomerase in senescing cells. A related aspect involvesscreening for a telomerase derepression agent by contacting a potentialagent with a cell lacking telomerase activity, and determining whetherthe agent increases the level of telomerase activity, e.g., by using acell expressing an inducible T antigen. Such an assay allows rapidscreening of agents which are present in combinatorial libraries, orknown to be carcinogens.

[0096] Applicant recognizes that known agents may be useful in treatmentof cancers since they are active at telomerase itself, or at the geneexpressing the telomerase. Thus, such agents can be identified in thisinvention as useful in the treatment of diseases or conditions for whichthey were not previously known to be efficacious. Indeed, agents whichwere previously thought to lack utility because they have little if anyeffect on cell viability after only 24-48 hours of treatment, can beshown to have utility if they are active on telomerase in vivo, and thusaffect cell viability only after several cell divisions.

[0097] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof, andfrom the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0098] The drawings will first briefly be described.

DRAWINGS

[0099] FIGS. 1-3 are graphs where the cell type and/or the cultureconditions are varied, plotting days in culture (horizontal axis) lengthversus cell number (vertical axis).

[0100]FIG. 4 is a linear plot of mean terminal restriction fragment(TRF) length versus PDL for human umbilical vein endothelial cellcultures. The plot had a slope (m) of −190±10 bp/PD, r=−0.98, P=0.01.

[0101]FIG. 5 is a plot of mean TRF of endothelial cell cultures fromhuman iliac arteries and iliac veins as a function of donor age.Parameters for iliac arteries are: m=−102 bp/yr, r=−0.98, P=0.01 and foriliac veins are: m=−42 bp/yr, r=−0.71, P=0.14.

[0102]FIG. 6 is a plot of decrease in mean TRF of medial tissue from theaortic arch, abdominal aorta, iliac artery and iliac vein as a functionof donor age. Parameters for linear plot are: m=−47 bp/yr, r=−0.85,P=0.05.

[0103]FIG. 7 is a plot of mean TRF length from PBLs plotted as afunction of donor age. The slope of the linear regression line (−41±2.6bp/y) is significantly different from 0 (p<0.00005).

[0104]FIG. 8 is a plot showing accelerated telomere loss in Down'sSyndrome (DS) patients. Genomic DNA isolated from PBLs of DS patientswas analyzed as described in FIG. 7. Mean TRF length is shown as afunction of donor age, for DS patients (open squares), and age-matchedcontrols (solid squares). The slope of the linear regression lines(−133±15 bp/y, trisomy, vs −43±7.7, normals) are significantly different(p<0.0005)

[0105]FIG. 9 is a plot showing decrease in mean TRF length in culturedT-lymphocytes as a function of population doubling (shown for DNA fromtwo normal individuals). Donor ages for these cells were not available.The slopes of these lines (−80±19 (°) and −102±5.4 (O) bp/doubling) aresignificantly different from zero (p<0.0001). Mean TRF length atterminal passage from a third donor for which multiple passages were notavailable is also shown (upsidedown V-symbol).

[0106]FIG. 10 is a copy of an autoradiogram showing TRF lengths ofovarian carcinoma and control normal cells. DNA from cells in asciticfluid from 2 patients (cas and wad) was digested with HinfI and RsaIseparated by electrophoresis, hybridized to the telomeric probe³²P(CCCTAA)₃, stringently washed and autoradiographed. The cells ofascitic fluid from 7 other patients were separated into adhering normalcells (N) and tumour clumps in the media (T). The DNA was extracted andrun as above. DNA from patient was obtained from both the first andforth paracentesis. Tumour cells from patients were cultured and DNA wasobtained at the respected population doubling (pd).

[0107]FIG. 11 shows telomerase activity in ovarian carcinoma cells. S100extracts from the previously studied transformant cell line 293 CSH, thetumor cell line HEY, purified tumour cell population and cells directlyfrom the ascitic fluid from patients were incubated with the telomereprimer (TTAGGG)₃ in the presence of DATP and TTP, ³²PdGTP and buffer.The reaction products were separated on a sequencing gel and exposed toa PhosphoImager screen. Either single (1) or double reactions (2) weretested.

[0108]FIG. 12 is a copy of an autoradiogram showing TRF lengths inHME-31 cells and HME31-E6 cells to extended lifespan (PD68) andsubsequent immortalization and stabilization of telomere length (PD81,107).

[0109]FIG. 13 is a copy of an autoradiogram showing the effect of CTO ontelomere length during the senescence of HME31:E6 cells. An intermediatetime point is chosen to show the dose-dependent protective effect of CTOoligonucleotide.

[0110]FIG. 14 is a graph showing extension of the life span of IMR90lung fibroblast cells in response to the CTO oligonucleotide.

[0111]FIGS. 15 and 16 are copies of autoradiograms showing the effect ofGTO on telomere length in IDH4 cells.

[0112]FIG. 17 is a graph showing extension of the life span of HME31:E6human breast epithelial cells in response to the CTO oligonucleotide.

[0113]FIG. 18A. shows the templating portion of the Tetrahymenatelomerase RNA with residues numbered 1 (5′) through 9 (3′) below it.The oligonucleotide primer with the sequence T₂G₄T₂G₄ binds to thetemplate by the base-pairing shown. Elongation followed by templatetranslocation are thought to occur as indicated.

[0114]FIG. 18B shows positions of major chain termination on thetelomerase RNA template by different nucleoside triphosphate analogs.The telomerase RNA template sequence is shown as in FIG. 18A. Arrowsindicate the position of maximal chain termination for each nucleosidetriphosphate (derived from the nucleoside) analog shown.

[0115]FIG. 19A-F are graphs showing that nucleoside analog triphosphatesinhibit incorporation of a ³²p label in a Tetrahymena telomerase assay.The effect of adding increasing concentrations of the analog, unlabeleddGTP or unlabeled TTP on the incorporation of labeled nucleotides wasmeasured using a quantitative telomerase reaction assay. Radioactivityincorporated (cpm) was plotted against the concentration of competitorsindicated in each panel. (A. labeled with [α-³²P]TTP. B-F. labeled with[α-³²P]dGTP. F. Effect of streptomycin sulfate on the telomerasereaction. The incorporation in the presence of 40 mM sodium sulfate isshown as the control for streptomycin sulfate).

[0116]FIG. 20A and B show the effect of nucleoside triphosphate analogson pausing patterns and processivity of telomerase in vitro.Specifically, FIG. 20A shows telomerase reactions in the presence orabsence of the indicated nucleoside triphosphate analogs. Unlabeled TTPcompetitor was also analyzed as a control, with and without primer inthe reaction mix. Products were then analyzed on a denaturingpolyacrylamide gel. FIG. 20B shows standard telomerase reactions wereperformed in the presence of ddGTP (lanes 4-6), ddITP (lanes 7-9), orDMSO (lane 1). DMSO was the solvent for ddGTP and at the highestconcentration tested (1%) showed no effect on the reactions comparedwith control reactions run without analog or DMSO (control lanes 2-3).Products were analyzed on a denaturing polyacrylamide gel.

[0117]FIG. 21A-D shows Southern blot analysis to demonstrate the effectof nucleoside analogs on telomere length in vivo, using anick-translated [α-³²P]-labeled plasmid containing a 3′ rDNA fragment asprobe. Genomic DNA was digested with PstI and BamHI and the rDNAtelomeres analyzed. The telomeric PstI fragment from the rDNA is betweenthe 1.6 and 1.0 kb markers, indicated as lines on both sides of eachpanel. The constant 2.8 kb band is the adjacent internal PstI rDNAfragment. Specifically, FIG. 21A shows results with a clone ofTetrahymena thermophila grown in 2% PPYS in the absence (−) and threeclones in the presence (+) of 5 mM AZT. Each set of three lanes showsthe results for a single cell clone grown vegetatively and transferredafter 3 days (lanes 1, 4, 7, 10), 10 days (lanes 2, 5, 8, 11) and 16days (lanes 3, 6, 9, 12). FIG. 21B shows that growth in differentconcentrations of AZT consistently resulted in concentration-dependentshortening of telomeres in log phase cells grown in thymine-deficientbroth (Isobroth) plus AZT. DNA made from cells sampled at 6, 10, and 16days show that shortened telomere lengths remain constant between 6 and16 days in culture. Lanes 1, 5, 9, 0 mM AZT control; lanes 2, 6, 10,0.01 mM AZT; lanes 3, 7, 11, 0.1 mM AZT; lanes 4, 8, 12, 1 mM AZT. FIG.21C shows cells grown vegetatively in 2% PPYS with no addition (lane 1),with 1% DMSO, the solvent for Ara-G, (“C”, lanes 2 and 5), and withAra-G (lane 3, 1 mM; lanes 4 and 6, 2 mM ) at 14 and 27 days in culture.FIG. 21D shows analysis of DNA from single-cell cultures grown inIsobroth plus 1 mm AZT (lanes 2 and 3) segregated into two classes basedon growth rate: “slow” (“S”, 0-1 doubling per day, lane 2) or “fast”(“F”, 2-4 doubling per day, lane 3). DNA from control cultures grown inthe absence of AZT are indicated (“C”, 2-4 doubling per day, lane 1).Several cultures were pooled in order to obtain sufficient DNA foranalysis.

[0118]FIG. 22 shows PCR analysis of DNA from Tetrahymena cellsconjugated in the presence of analog and starved for the duration ofmating. A Telomeric a primer and a 5′ rDNA primer were used in PCRreactions with DNA from cells conjugated in the presence or absence ofanalog to detect the addition of telomeres to the 11 Kb rDNA formedduring macronuclear development. A reaction was run without DNA as acontrol. Tests included use of 5 mM AZT; 1 mM Ara-G, and 1 mM Acyclo-G.SB210 cells were also mock-conjugated as a control. The expected productis approximately 1400 bp. In addition, 3′ micronuclear rDNA primers wereused on the same DNA to demonstrate the presence and competence of theDNA samples for PCR. The expected band is 810 bp. In the figure southernblot analysis of the 5′ rDNA telomeric PCR reactions using arandom-primed ³²P-labeled 5′rDNA probe confirmed the 1400 bp PCR productas part of the 5′rDNA with telomeres, from the 11 Kb rDNA species formedtransiently during macronuclear development. No hybridization is seen inthe no DNA control (lane 1) or the SB210 mock-conjugated control (lane6). Lane 2, no added analog; lane 3, 5 mM AZT, lane 4, 1 mM Ara-G; lane5, 1 mM Acyclo-G; lane 6, mock-conjugated SB210 cell DNA. These resultswere reproduced in three separate experiments.

[0119]FIG. 23 shows growth of cultured JY lymphoma cells with RPMImedium and no added agents (control) and with a relatively low dose ofddG, AZT, ara-G, and ddI. The DMSO is a control for ddG.

[0120]FIG. 24 shows the growth of cultured JY lymphoma cells cultured inan analogous manner to those in FIG. 23, but treated with relativelyhigher doses of potential telomerase inhibitors.

[0121]FIG. 25 shows Southern blot of DNA isolated from JY lymphoma cellsat weeks one and three probed with the telomeric repeat sequence(TTAGGG)₃. The first lane is DNA from the cells at the start of theexperiment, the second is the RPMI control, and the third is cellstreated with AZT for the times indicated.

[0122]FIG. 26 shows fibroblast DNA hybridized by Southern blot to thetelomeric (TTAGGG)₃ probe. Lane labeled “HinfI” is DNA digested with therestriction enzyme HinfI, the lane labeled “O” had no treatment, thelane labeled “P only” was treated with piperidine, and the lane labelled“P+DMS” was piperidine and dimethyl sulfate treated.

[0123]FIG. 27 shows the inhibition of human telomerase achieved by theagent ddG at various dosages in three separate experiments. Thetelomerase was derived from the tumor cell line 293.

[0124]FIG. 28 shows hybridization of C. albicans telomeric repeats togenomic DNAs of a variety of other Candida species. Genomic DNAs ofeight species of yeasts were digested with EcoRI, electrophoresed on0.8% agarose, blotted, and then probed with a ³²P-labeled telomericfragment from C. albicans WO-1. Hybridization was carried out at 55° C.and washes were at the same temperature in Na₂HPO₄ at 200 mM Na+ and 2%SDS. DNA size markers, measured in kilobase pairs (kb), are shown at theright. The species used here are C. quillermondii, S. cerevisiae, C.pseudotropicalis, Kluyveromyces lactis, C. lusitaniae, C. maltosa, C.tropicalis, and C. albicans. Asterisks indicate particular strains fromwhich telomeres were cloned. Strains beginning with “B” are N.I.H.strains obtained from B. Wickes.

[0125]FIG. 29 shows Bal31 sensitivity of genomic copies of the tandemrepeats in K. lactis ATCC 32143 (left panel) and C. guillermondii B-3163(right panel). Uncut yeast genomic DNAs were incubated with Bal3lnuclease for increasing periods of time (given in minutes above eachlane), then digested with EcoRI and electrophoresed on a 0.8% agarosegel, and blotted onto a nylon membrane. For K. lactis, probing was donewith a ³²P-kinased 25 base oligonucleotide identical in sequence to theK. lactis telomeric repeat shown in FIG. 30. Hybridization and washeswere carried out at 49° C. For C. guillermondii, probing was done with³²P-labeled pCgui3, a pBluescript vector (Stratagene, LaJolla, Calif.)carrying a α-2 kb telomeric clone from C. guillermondii. Hybridizationand washing (in 200 mM Na⁺) were carried out at 54° C. Most bands aregone by the 1 min. time point. Approximately three other bands areshortening but are not gone at 3 min. These latter bands presumably arehomologous to the particular subtelomeric sequences present in pCgui3.DNA size markers (in kb) are indicated at the right of each panel.

[0126]FIG. 30 shows sequences of telomeric repeats from several buddingyeast species. Specifically, telomere-enriched libraries wereconstructed from genomic DNA by standard methods. Uncut yeast genomicDNA was ligated to a blunt-ended linearized plasmid vector and then thisligated mix was digested with a restriction enzyme that cleaves bothwithin the vector's polylinker and within a few kilobases of at leastsome of the putative telomeric ends of the species in question. Noenzymatic pre-treatment was done to produce blunt-ends of the telomeresin the genomic DNA prior to the initial ligations. Plasmids were thenrecircularized with T4DNA ligase, and transformed into E. coli cellsprior to screening for putative telomere clones by colony hybridization.The libraries from C. maltosa, C. pseudotropicalis, two strains of C.tropicalis, and K. lactis ATCC 32143, species which showed multiplebands that cross hybridized to the C. albicans telomeric repeat probe,were screened with this probe. A cloned S. cerevisiae telomere probe(repeat unit TG₂₋₃(GT)₁₋₃) was used to screen the telomere-enrichedlibrary from C. glabrata, whose genomic DNA cross-hybridized with this,but not with the C. albicans telomeric repeat probe. C. guillermondiiDNA did not appreciably cross-hybridize with either the C. albicans orthe S. cerevisiae telomeric probes at the stringencies tested. Thetelomere-enriched library from this species was screened using totalgenomic C. guillermondii DNA as a probe. This procedure can be used toidentify all clones containing repetitive sequences and we reasoned thattelomeres should be a reasonable percentage of the repetitive sequencesfound in telomere enriched libraries. Typically, a few hundred E. colitransformants were obtained for each small library and up to nineputative telomere clones were obtained from each. Nine repetitive DNAclones were obtained from C. guillermondii, three of which proved to betelomeric.

[0127]FIG. 31 shows two types of telomeric repeats present in certain C.tropicalis strains. Genomic DNAs from ten (only five here are shown) C.tropicalis strains and C. albicans WO-1 were digested with ClaI,eletrophoresed on a 0.8% agarose gel, blotted, and probed witholigonucleotides specific to either the “AC form of C. tropicalistelomeric repeat (left panel) or to the “AA” form of repeat (rightpanel). Sequences of these two oligonucleotides are: 5′ACGGATGTCACG(“AC”) and 5′GTGTAAGGATG (“AA”) with the position of the dimorphic baseshown underlined. Hybridization with the kinased “AC” probe was at 47°C., and hybridization with the “AA” probe at 24° C. Washes for both werein 2% SDS with 500 mM Na⁺. The specificity of the “AA” probe isindicated by its failure to hybridize with the C. albicans telomeres,despite only one base mismatch and the fact that the C. albicans cellsused here have much longer telomeres (and therefore many more telomericrepeats) than do C. tropicalis strains. The shortness of the C.tropicalis telomeres may explain why they appear to be particularlyhomogeneous in size, as is suggested by the relative sharpness ofindividual telomeric bands.

[0128]FIG. 32 shows a Southern blot of DNA isolated from JY cellshybridized to the (TTAGGG)₃ probe. Cells were treated over a 10 weekperiod with either 10 μM ddG in 0.01% DMSO or medium with 0.01% DMSOonly. Cells treated with ddG showed a marked decrease in mean telomerelength consistant with the inhibition of telomerase activity.

[0129]FIG. 33 shows telomerase activity in cells from ascitic fluid.Specifically, S100 extracts were prepared, protein concentrationsdetermined and telomerase activity assayed by incubating S100 extractswith an equal volume of reaction mixture containing buffer, telomereprimer (TTAGGG)₃, α³²PdGTP, TTP and DATP, at 30° C. for 1 hour. Thereactions were terminated with RNase followed by deproteination withproteinase K. Unincorporated α³²PdGTP was removed using NICK SPINcolumns (Pharmacia) according to the supplier's direction. Products wereresolved on a sequencing gel and exposed to either a PhosphorImagerscreen (Molecular Dynamics). A ladder (L) and kinased 5′³²P(TTAGGG)₃ (O)were run as markers. FIG. 33A shows telemerase assayed in S100 extractswith equal protein concentration (≈11 mg/ml) prepared from the controlhuman cell line 293 CSH, a subline of 293 cell line, and fromunfractionated ascitic fluid cells from patient Dem-1 and Rud-1. Inlanes 1, 3 and 5 RNase was added to the extracts prior to addition ofα³²PdGTP. FIG. 33B shows S100 extracts isolated and assayed fortelomerase activity from the early passage cultures of cells frompatients Pres-3 and Nag-1 compared to 293 cells. All extracts wereassayed at a protein concentration of ≈2-3 mg/ml.

TELOMERES AND TELOMERASE

[0130] All normal diploid vertebrate cells have a limited capacity toproliferate, a phenomenon that has come to be known as the Hayflicklimit or replicative senescence. In human fibroblasts, this limit occursafter 50-100 population doublings, after which the cells remain in aviable but non-dividing senescent state for many months. This contraststo the behavior of most cancer cells, which have escaped from thecontrols limiting their proliferative capacity and are effectivelyimmortal.

[0131] One hypothesis to explain the cause of cellular senescenceconcerns the role of the distal ends of chromosomes called telomeres.The hypothesis is that somatic cells lack the ability to replicate thevery ends of DNA molecules. This results in a progressive shortening ofthe ends of the chromosomes until some function changes, at which timethe cell loses the capacity to proliferate.

[0132] DNA polymerase synthesizes DNA in a 5′ to 3′ direction andrequires a primer to initiate synthesis. Because of this, the “laggingstrand” does not replicate to the very ends of linear chromosomes. Thechromosome is thus shortened with every cell division. The ends ofchromosomes are called telomeres, and are composed of long TTAGGGrepeats. The enzyme telomerase can add TTAGGG repeats to the 3′ end ofthe telomeric DNA, thus extending the DNA and preventing shortening.

[0133] Germline cells have long telomeres and active telomerase. Somaticcells lack telomerase activity, and their telomeres have been found toshorten with cell division both in vivo and in culture. Cancer cells areimmortal, and have regained telomerase activity and thus can maintaintheir chromosome ends. Examples are provided below of definitiveexperiments which indicate that telomere shortening and telomeraseactivity are key factors in controlling cellular senescence andimmortalization.

[0134] Methods

[0135] As noted above, the present invention concerns diagnosis andtherapy associated with measuring telomeric length and manipulatingtelomerase-dependent extension or telomerase-independent shortening.While the invention is directed to humans, it may be applied to otheranimals, particularly mammals, such as other primates, and domesticanimals, such as equine, bovine, avian, ovine, porcine, feline, andcanine. The invention may be used in both therapy and diagnosis. In thiscase of therapy, for example, telomere shortening may be slowed orinhibited by providing DNA oligonucleotides by reactivating orintroducing telomerase activity, or their functional equivalent, orindefinite proliferation can be reduced by inhibiting telomerase. In thecase of diagnostics, one may detect the length of telomeres as to aparticular chromosome or group of chromosomes, or the average length oftelomeres. Diagnosis may also be associated with determining theactivity of telomerase, or the presense of the components of the enzymeeither on a protein or RNA level, in cells, tissue, and the like.

[0136] Information on the relative age, remaining proliferativecapacity, as well as other cellular characteristics associated withtelomere and telomerase status may be obtained with a wide variety ofcell types and tissues, such as embryonic cells, other stem cells,somatic cells (such as hepatocytes in the context of cirrhosis),connective tissue cells (such as fibroblasts, chondrocytes, andosteoblasts), vascular cells (such as endothelial and smooth musclecells), cells located in the central nervous system (such as brainastrocytes), and different neoplastic tissues, and parasitic pathogenswhere it is desirable to determine both the remaining replicativecapacity of the hyperplastic cells and their capacity for immortalgrowth to predict growth potential.

[0137] Maintaining Telomere Length

[0138] Telomere length in cells in vitro or in vivo may be usefullymaintained by a variety of procedures. These include those methodsexemplified below. These examples, however, are not limiting in thisinvention since those in the art will recognize equivalent methods. Itis expected that all the methods will be useful in manipulating telomerelength now that applicant has demonstrated this experimentally. Suchmethods may be based upon provision of oligonucleotides or other agentswhich interact with telomeres to prevent shortening during celldivision. In addition, the methods include treatment with agents whichwill include telomerase, or its equivalent activity, within a cell toprevent shortening. Finally, the methods also include modulation of geneexpression associated with cell senescence.

[0139] Useful agents can be determined by routine screening procedures.For example, by screening agents which interact in an in vitro systemwith telomeres, and block loss of telomere ends, or aid increase intelomere length. Non-limiting examples of such methods are providedbelow. All that is necessary is an assay to determine whether telomereend shortening is reduced during cell division. The mechanism by whichsuch agents act need not be known, so long as the desired outcome isachieved. However, by identifying useful target genes (e.g., the M2mortality modulation gene(s)), antisense and equivalent procedures canbe designed to more appropriately cause desired gene expression ornon-expression (e.g., the de-repression of telomerase).

[0140] In a particular example (non-limiting in this invention) one canreduce the rate of telomere shortening, by providing a nucleic acid,e.g., DNA or RNA (including modified forms), as a primer to the cells.Such nucleic acid will usually include 2 to 3 repeats, more usually 2repeats, where the repeats are complementary to the G-rich DNA telomerestrand. Such oligonucleotides may be used to extend the proliferativecapability of cells.

[0141] The oligonucleotides can be transferred into the cytoplasm,either spontaneously (i.e., without specific modification) or by the useof liposomes which fuse with the cellular membrane, or are endocytosedby employing ligands which bind to surface membrane protein receptors ofthe cell resulting in endocytosis. Alternatively, the cells may bepermeabilized to enhance transport of the oligonucleotides into thecell, without injuring the host cells. Another way is to use a DNAbinding protein, e.g., HBGF-1, which is known to transport anoligonucleotide into a cell. In this manner, one may substantiallyreduce the rate of telomere shortening from an average of about 50 bpper division, to an average of about 6-12 bp per division (see examplesbelow), thus significantly extending the number of divisions occurringbefore induced cellular senescence.

[0142] By “senescence” is meant the loss of ability of a cell toreplicate in the presence of normally appropriate replicative signals,and may be associated with the expression of degradative enzymes, suchas collagenase. The term does not include quiescent cells which might beinduced to replicate under appropriate conditions. This term isexemplified below in the examples, where the number of cell doublingprior to senescence is increased.

[0143] The above processes are useful in vivo. As already indicated, byusing liposomes, particularly where the liposome surface carries ligandsspecific for target cells, or the liposomes will be preferentiallydirected to a specific organ, one may provide for the introduction ofthe oligonucleotides into the target cells in vivo. For instance,utilizing lipocortin affinity for phosphatidyl serine, which is releasedfrom injured vascular endothelial cells, the oligonucleotides may bedirected to such site. Alternatively, catheters, syringes, depots or thelike may be used to provide high localized concentrations. Theintroduction of such oligonucleotides into cells resulting in decreasedsenescence in response to cell division can have therapeutic effect.

[0144] The maintenance of telomere length has application in tissueculture techniques to delay the onset of cellular senescence. Forinstance, cell-based therapies which require the clonal expansion ofcells for reintroduction into an autologous patient are limited to about20-30 doublings. This invention allows, the expansion of cells in thecase of gene therapy, both prior to genetic manipulation and thenexpansion of the manipulated cells, the maintenance of telomere length.This in turn allows normal cells to be cultivated for extended doublingsin vitro. Experiments described below demonstrate the utility of thismethod in vitro, and demonstrate its applicability in vivo.

[0145] Critical shortening of telomeres leads to a phenomenon termed“crisis” or M2 senescence. See, Shay et al., 1992, supra. Among thecells in crisis, rare mutants may become immortalized in which M2 geneshave altered regulation, and where expression of telomerase isreactivated and stabilizes the telomere length. An M2 regulatory genemay be modulated to provide a useful means of modulating telomere lengthand telomerase activity. The M2 genes may be identified by means ofinsertional mutagenesis into cells in M2 crisis utilizing a retrovirus.Cells wherein the M2 gene has been knocked out will then grow inresponse to the reactivation of telomerase, and such cells can supply asource or DNA from which to clone the M2 genes. This technique hasyielded numerous cell clones in which the retrovirus has inserted into acommon restriction fragment. The repression of the M2 regulatory gene(s)by antisense or other means can provide a means of activating telomerasereversibly, such that telomeres may be extended and then telomeraseagain repressed. In this manner, proliferative capacity may be extendedwith or without the addition of oligonucleotides to slow the telomereshortening. Such cells may then be used in cell-based therapies, such asbone marrow transplantation, reconstitution of connective tissue, andtransplantation of early passage adrenal cortical cells, fibroblasts,epithelial cells, and myoblasts.

[0146] Telomerase Modulation

[0147] As discussed above, cancer cells contain telomerase activity andare thereby immortal. In addition, numerous types of parasitic pathogensare immortal and have active telomerase. Thus, it is useful to modulate(e.g., decrease) telomerase activity in such cells to impart a finitereplicative life span. In contrast to the long telomeric tracts innormal human cells, tracts of telomeric DNA in protozoan cells, fungalcells, and some parasitic worms, as well as many cancer cells, aretypically shorter. This makes these cells more vulnerable to telomeraseinhibitors than normal human cells (e.g. germ line cells).

[0148] Thus, inhibition or induction of telomerase has applications invarious situations. By inhibiting telomerase intracellularly, one mayreduce the ability of cancer cells to proliferate. Telomerase may becompetitively inhibited by adding synthetic agents, e.g.,oligonucleotides comprising 2 or more, usually not more than about 50repeats, of the telomeric motif of the 5′-3′ G-rich strand (the strandwhich acts as the template). The oligonucleotides may be synthesizedfrom natural or unnatural units, e.g., the derivatives or carbonderivatives, where a phosphate-oxygen is substituted with sulfur ormethylene, modified sugars, e.g., arabinose, or the like. As discussedabove, other equivalent agents may also be used to inhibit or causeexpression of telomerase activity.

[0149] The oligonucleotides may be introduced as described above so asto induce senescence in the immortalized cells, in culture and in vivo.Where growing cells in culture, where one wishes to prevent immortalizedcells from overgrowing the culture, one may use the subjectoligonucleotides to reduce the probability of such overgrowth. Thus, bymaintaining the oligonucleotides in the medium, they will be taken up bythe cells and inhibit telomerase activity. One may provide for linkageto the telomeric sequence with a metal chelate, which results incleavage of nucleic acid sequences. Thus, by providing iron chelatebound to the telomeric motif, the telomerase RNA will be cleaved, so asto be non-functional. Alternatively, a reactive group may be coupled tothe oligonucleotide that will covalently bind to telomerase, or the 3′residue may be made to be dideoxy so as to force chain termination.

[0150] Alternatively, one may introduce a ribozyme, having 5′ and3′-terminal sequences complementary to the telomerase RNA, so as toprovide for cleavage of the RNA. In this way, the telomerase activitymay be substantially inhibited, so as to result in a significantlimitation of the ability of the cancer cells to proliferate. Telomerasemay also be inhibited by the administration of an M2 regulator geneproduct. By modulating the expression of any of the proteins directlyregulating telomerase expression, one may also modulate cellulartelomerase activity.

[0151] Alternatively, one may use a screening assay utilizing human ortetrahymena telomerase to screen small molecules e.g., nucleosideanalogs like ava-G, ddG, AZT, and the like and RNA and DNA processingenzyme inhibitors, alkylating agents, and various potential anti-tumordrugs. These may then be further modified.

[0152] The nucleic acid sequences may be introduced into the cells asdescribed previously. Various techniques exist to allow for depotsassociated with tumors. Thus, the inhibiting agents or nucleic acids maybe administered as drugs, since they will only be effective only incells which include telomerase. Since for the most part, human somaticcells lack telomerase activity they will be unaffected. Some care may berequired to prevent entry of such drugs into germ cells, which mayexpress telomerase activity.

[0153] The subject compositions can therefore be used in the treatmentof neoplasia wherein the tumor cells have acquired an immortal phenotypethrough the inappropriate activation of telomerase, as well as varioushuman and veterinary parasitic diseases; including human protozoalpathogens such as; amebiasis from Entamoeba histolytica, amebicmeningoencephalitis from the genus Naegleria or Acanthamoeba, malariafrom Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, andPlasmodium falciparum, Leishmaniasis from such protozoa as Leishmaniadonovani, Leishmania infantum, Leishmania chagasi, Leishmania tropica,Leishmania major, Leishmania aethiopica, Leishmania mexicana, andLeishmania braziliensis, Chagas' disease from the protozoan Trypanosomacruzi, sleeping sickness from Trypanosoma brucei, Trypanosoma gambiense,and Trypanosoma rhodesiense, Toxoplasmosis from Toxoplasma gondii,giardiasis from Giardia lamblia, cryptosporidiosis from Cryptosporidiumparvum, trichomoniasis from Trichomonas vaginalis, Trichomonas tenax,Trichomonas hominis, pneumocystis pneumonia from Pneumocystis carinii,bambesosis from Bambesia microti, Bambesia divergens, and Bambesiaboris, and other protozoans causing intestinal disorders such asBalantidium coli and Isospora belli. Telomerase inhibitors would also beuseful in treating certain helminthic infections including the species:Taenia solium, Taenia saginata, Diphyllobothrium lata, Echinococcusgranulosus, Echinococcus multilocularis, Hymenolepis nana, Schistosomamansomi, Schistosoma japonicum, Schistosoma hematobium, Clonorchissinensis, Paragonimus westermani, Fasciola hepatica, Fasciolopsis buski,Heterophyes heterophyes, Enterobius vermicularis, Trichuris trichiura,Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus,Strongyloides stercoralis, Trichinella spiralis, Wuchereria bancrofti,Onchocerca volvulus, Loa loa, Dracunculus medinensis, and fungalpathogens such as: Sporothrix schenckii, Coccidioides immitis,Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioidesbrasiliensis, Candida albicans, Cryptococcus neoformans, Aspergillusfumigatus, Aspergillus flavus, fungi of the genera Mucor and Rhizopus,and species causing chromomycosis such as those of the generaPhialophora and Cladosporium, and important veterinary protozoalpathogens such as: Babesia caballi, Babesia canis, Babesia egui, Babesiafelis, Balantidium coli, Besnoitia darlingi, Eimeria acervulina, Eimeriaadenoeides, Eimeria ahsata, Eimeria alabamensis, Eimeria auburnensis,Eimeria bovis, Eimeria brasiliensis, Eimeria brunetti, Eimeriacanadensis, Eimeria cerdonis, Eimeria crandallis, Eimeria cylindrica,Eimeria debliecki, Eimeria despersa, Eimeria ellipsoidalis, Eimeriafauvei, Eimeria gallopavonis, Eimeria gilruthi, Eimeria granulosa,Eimeria hagani, Eimeria illinoisensis, Eimeria innocua, Eimeriaintricata, Elmeria leuskarti, Eimeria maxima, Eimera meleagridis,Eimeria meleagrimitis, Eimeria mitis, Eimeria mivati, Eimeria necatrix,Eimeria neodebliecki, Eimeria ninakohlyakimorae, Eimeria ovina, Eimeriapallida, Eimeria parva, Eimeria perminuta, Eimeria porci, Eimeriapraecox, Eimeria punctata, Eimeria scabra, Eimeria spinoza, Eimeriasubrotunda, Eimeria subsherica, Eimeria suis, Eimeria tenella, Eimeriawyomingensis, Eimeria zuernii, Endolimax gregariniformis, Endolimaxnana, Entamoeba bovis, Entamoeba gallinarum, Entamoeba histolytica,Entamoeba suis, Ciardia bovis, Giardia canis, Giardia cati, Giardialamblia, Haemoproteus meleagridis, Hexamita meleagridis, Histomonasmeleagridis, Iodamoeba buetschili, Isospora bahiensis, Isosporaburrowsi, Isospora canis, Isospora felis, Isospora ohioensis, Isosporarivolta, Isospora suis, Klossiella equi, Leucocytozoon caallergi,Leucocytozoon smithi, Parahistomonas wenrichi, Pentatrichomonas hominis,Sarcocystis betrami, Sarcocystis bigemina, Sarcocystis cruzi,Sarcocystis fayevi, hemionilatrantis, Sarcocystis hirsuta, Sarcocystismiescheviana, Sarcocystis muris, Sarcocystis ovicanis, Sarcocystistenella, Tetratrichomonas buttreyi, Tetratrichomonas gallinarum,Theileria mutans, Toxoplasma gondii, Toxoplasma hammondi, Trichomonascanistomae, Trichomonas gallinae, Trichomonas felistomae, Trichomonaseberthi, Trichomonas equi, Trichomonas foetus, Trichomonas ovis,Trichomonas rotunda, Trichomonas suis, and Trypanosoma melophagium. Inaddition, they can be used for studying cell senescence, the role oftelomeres in the differentiation and maturation of cells from atotipotent stem cell, e.g., embryonic stem cells, or the like, and therole of telomerase in spermatogenesis.

[0154] Telomere Length

[0155] Procedures for measuring telomere length are known in the art andcan be used in this invention. Typically, restriction endonucleasedigestion is used (with enzymes which do not cleave telomeric DNA), andthe length of the fragment having detectable telomere DNA is separatedaccording to molecular weight by agarose gel electrophoresis. Given thatthe DNA sequence of a telomere is known, detection of such DNA isrelatively easy by use of specific oligonucleotides. Examples of thesemethods are provided below.

[0156] For diagnosis, in detection of the telomeric length, one maystudy just a particular cell type, all cells in a tissue (where variouscells may be present), or subsets of cell types, and the like. Thepreparation of the DNA having such telomeres may be varied, dependingupon how the telomeric length is to be determined.

[0157] Conveniently, the DNA may be isolated in accordance with anyconventional manner, freeing the DNA of proteins by extraction, followedby precipitation. Whole genomic DNA may then be melted by heating to atleast about 80° C., usually at least about 94° C., or using high saltcontent with chaotropic ions, such as 6×SSC, quanidinium thiocyanate,urea, and the like. Depending upon the nature of the melting process,the medium may then be changed to a medium which allows for DNAsynthesis.

[0158] (a) DNA Synthesis

[0159] In one method, a primer is used having at least about 2 repeats,preferably at least about 3 repeats of the telomeric sequence, generallynot more than about 8 repeats, conveniently not more than about 6repeats. The primer is added to the genomic DNA in the presence of only3 of the 4 nucleoside triphosphates (having the complementarynucleosides to the protruding or G-rich strand of a telomere, e.g., A, Tand C for human chromosomes), DATP, dTTP and dCTP. Usually at least theprimer or at least one of the triphosphates is labeled with a detectablelabel, e.g., a radioisotope, which label is retained upon incorporationin the chain. If no label is used, other methods can be used to detectDNA synthesis. The primer is extended by means of a DNA polymerase,e.g., the Klenow fragment of DNA polymerase I, T7 DNA polymerase or TaqDNA polymerase

[0160] The length of the extended DNA can then be determined by varioustechniques, e.g., those which separate synthesized DNA on the basis ofits molecular weight, e.g., gel electrophoresis. The DNA synthesized maythen be detected based on the label, e.g., counts incorporated per μg ofDNA, where the counts will be directly proportional to telomere length.Thus, the measure of radioactivity in relation to the amount of DNA willsuffice to quantitate telomere length.

[0161] If desired, telomeres of known length may be used as standards,whereby a determination of radioactivity may be read off a standardcurve as related to telomere length. Instead, one may prepare tissueswhere individual cells may be assayed for relative telomere length by insitu hybridization. In this approach, for example, the primer is labeledwith a detectable label, usually biotin or digoxygenin. Followingannealing to prepared tissue sections or cells, the label is revealedhistochemically, usually using autoradiography (if the label wereradioactive), using avidin/streptavidin (if the label were biotin) orusing antidigoxygenin antibodies (if the label were digoxygenin). Theamount of signal per cell is proportional to the number of telomericrepeats, and thus to the telomere length. This can be quantitated bymicrofluorometry or analogous means, and compared to the signal fromstandard cells of known telomere length to determine the telomere lengthin the test sample.

[0162] (b) Restriction Endonuclease Digestion

[0163] Alternatively, one may use primers which cause covalentcross-linking of the primer to telomere DNA. In this situation, one maytotally digest the DNA with restriction endonucleases which have 4 baserecognition sites, which results in the production of relatively shortfragments of DNA, except for telomeric DNA which lacks the recognitionsite. Restriction endonucleases which may find use include AluI, HinfI,MspI, RsaI, and Sau3A, where the restriction endonucleases may be usedindividually or in combination. After digestion of the genomic DNA, theprimer may be added under hybridizing conditions, so as to bind to theprotruding chain of the telomeric sequence. By providing for twomoieties bound to the primer, one for covalent bonding to the telomericsequence and the other for complex formation with a specific bindingpair member, one can then provide for linking of a telomeric sequence toa surface. For example, for covalent bonding to the telomeric sequence,psoralen, or isopsoralen, may be linked to one of the nucleotides by abond or chain and upon UV-radiation, will form a bridge between theprimer and the telomere.

[0164] The specific binding pair member will normally be a hapten, whichbinds to an appropriate complementary member, e.g., biotin andstrept/avidin, trinitrobenzoic acid and anti-trinitrobenzamide antibody,or methotrexate and dihydrofolate reductase. Rather than having themoiety for covalent bonding covalently bonded to the primer, one may adda compound into the medium which is intercalatable into the nucleicacid, so as to intercalate between double-stranded nucleic acidsequences. In this manner, one may achieve the same purpose. Use of asubstantial excess of the intercalatable compound will cause it to alsointercalate into other portions of DNA which are present. Variousmodifications of this process may be achieved, such as size separation,to reduce the amount of label containing DNA.

[0165] The specific binding pair member may be used for separation oftelomeric DNA free of contaminating DNA by binding to the complementarypair member, which may be present on beads, on particles in a column, orthe like. In accordance with the nature of the separation, thecovalently bonded telomere strand may now be purified and measured forsize or molecular weight. Again, if desired, standards may be employedfor comparison of distribution values.

[0166] The specific binding pair member hapten can be present at the5′-terminus of the primer or at intermediate nucleotides. Specifically,biotin-conjugated nucleotides are generally available and may be readilyintroduced into synthetic primer sequences in accordance with knownways.

[0167] The above-described techniques can also be used for isolating andidentifying DNA contiguous to the telomere.

[0168] (c) Average Telomere Length

[0169] In methods of this invention it may be useful to determineaverage telomere length by binding a primer to a telomere prior toseparation of the telomeric portion of the chromosomes from other partsof the chromosomes. This provides a double-stranded telomeric DNAcomprising the telomeric overhang and the primer. A reaction may then becarried out which allows for specific identification of the telomericDNA, as compared to the other DNA present. The reaction may involveextension of the primer with only 3 of the nucleotides (dNTPs), using alabeled nucleotide, covalent bonding of the primer to the telomericsequence, or other methods which allow for separation of the telomericsequence from other sequences. The length of the synthesized DNAdetected then represents the average telomere length.

[0170] Telomere length can also be measured directly by the “anchoredterminal primer” method. In this method, the 3′ ends of genomic DNA arefirst “tailed” with dG nucleotides using terminal transferase.Telomeres, which are known to have 3′ overhangs, then would have one ofthe three following conformations:

[0171] . . . 5′TTAGGGTTAGGGTTAGGGGGGGGGGG . . . 3′

[0172] . . . 5′TTAGGGTTAGGGTTGGGGGGGGGGGG . . . 3′

[0173] . . . 5′TTAGGGTTAGGGTGGGGGGGGGGGGG . . . 3′

[0174] Other ends of the genomic DNA which were generated by shearingwould be tailed with G's but would not have the adjacent TTAGGG repeats.Thus, a mix of the following 3 biotinylated oligonucleotides wouldanneal under stringent conditions specifically to all possible telomereends:

[0175] 5′B-CCCCCCCCTAACCCTA

[0176] 5′B-CCCCCCCCAACCCTAA oligo Mix [M]

[0177] 5′B-CCCCCCCCACCCTAAC

[0178] Oligo mix [M] consists of 16-base oligonucleotides with 5′ biotin(B), but other combinations of 5′-C-tracts adjacent to the C-richtelomeric repeats could provide specific hybridization to the 3′ end ofthe native telomeres.

[0179] Extension of the primer with a DNA polymerase such as Klenow, DNAPolymerase I, or Taq polymerase, in the presence of dCTP, dATP, dTTP (nodGTP, and with or without ddGTP) would stabilize the primer-templateconfiguration and allow selection, using streptavadin beads, of theterminal fragments of DNA containing the telomeric DNA. The length ofprimer extension using Klenow (monitored with labeled nucleotides) wouldindicate the length of the telomeric (GTR) 3′ overhang, since Klenowlacks 5′-3′ exonuclease activity and would stall ac the CTR. This lengthdistribution could be indicative of the level of telomerase activity intelomerase-positive cells (i.e., longer extensions correspond to greatertelomerase activity). In contrast, extension of the primer with DNApolymerase I, an enzyme with 5′-3′ exonuclease activity as well aspolymerase activity, would allow extension through the CTR until C's areencountered in the template strand (subtelomeric to the GTR). The lengthdistribution of this reaction, monitored by labeled nucleotides, wouldbe indicative of the length distribution of the GTR. In both cases,labeled products arising from biotinylated primers are selected with thestreptavadin beads to reduce the signal from non-specific priming.Alternatively, re-priming and extension of the tailed chromosome end cantake place after selection of the partially extended products with thestreptavadin beads, and after denaturation of the C-rich strand from theduplex.

[0180] Experiments have confirmed that the G-tailing of chromosome endscan be carried out efficiently such that about 50 G residues are addedper end, that the priming with the junction oligonucleotide mix ishighly specific for the tailed telomeric ends, and that streptavadinbeads select specifically for the extension products that originate fromthe biotinylated primers and not from other fortuitous priming events.The length of the extension products under the conditions outlined abovethus provide a direct estimate of the length of the terminal TTAGGGrepeat tract. This information is especially important in cases wherestretches of TTAGGG repeats occur close to but not at the termini ofchromosomes. No other method described to date is capable ofdistinguishing between the truly terminal TTAGGG repeats and suchinternal repeats.

[0181] The determination of telomere length as described above can beassociated with a variety of conditions of diagnostic interest.Following telomere length in tumor cells provides information regardingthe proliferative capacity of such cells before and followingadministration of inhibitors of telomerase (or other treatments whichdestabilizes the telomere length as discussed above). It also provides ameans of following the efficacy of any treatment and providing aprognosis of the course of the disease.

[0182] Where diseased tissue is involved, the native tissue can beevaluated as to proliferative capability. By “proliferative capability”is meant the inherent ability of a cell or cells in a tissue to dividefor a fixed number of divisions under normal proliferation conditions.That is, the “Hayflick” number of divisions, exemplified below in theexamples. Thus, despite the fact that the tissue may have a spectrum ofcells of different proliferative capability, the average value will beinformative of the state of the tissue generally. One may take a biopsyof the tissue and determine the average telomeric length. Using thevalue, one may then compare the value to average normal healthy tissueas to proliferative capability, particularly where the tissue iscompared to other tissue of similar age.

[0183] In cases of cellular diseases, such as liver disease, e.g.,cirrhosis, or muscle disease, e.g., muscular dystrophy, knowledge of theproliferative capability can be useful in diagnosing the likelyrecuperative capability of the patient. Other situations involve injuryto tissue, such as in surgery, wounds, burns, and the like, where theability of fibroblasts to regenerate the tissue will be of interest.Similarly, in the case of loss of bone, osteoarthritis, or otherdiseases requiring reformation of bone, renewal capability ofosteoblasts and chondrocytes will be of interest.

[0184] While methods are described herein to evaluate the proliferativecapacity of a tissue by taking an average measure of telomere length itis noted that the tissue may have a spectrum of cells of differentproliferative capability. Indeed, many tissues, including liver,regenerate from only a small number of stem cells (less than a fewpercent of total cells). Therefore, it is useful in this invention touse in situ hybridization (such as with fluorescently labeled telomericprobes), to identify and quantitate such stem cells, and/or thetelomeric status of such cells on an individual, rather than collectivebasis. This is performed by measuring the fluorescent intensity for eachindividual cell nucleus using, e.g., automated microscopy imagingapparatus. In addition to in situ hybridization, gel electrophoresis isuseful in conjunction with autoradiography to determine not only theaverage telomere length in cells in a tissue sample, but also thelongest telomere lengths (possibly indicating the presence of stemcells) and the size distribution of telomere lengths (which may reflectdifferent histological cell types within a tissue, see FIGS. 10-11).Thus, the autoradiogram, or its equivalent provides useful informationas to the total telomere status of a cell, or group of cells. Eachsegment of such information is useful in diagnostic procedures of thisinvention.

[0185] d) Modified Maxam-Gilbert Reaction

[0186] The most common technique currently used to measure telomerelength is to digest the genomic DNA with a restriction enzyme with afour-base recognition sequence like HinfI, electrophorese the DNA andperform a Southern blot hybridizing the DNA to a radiolabeled (TTAGGG)₃probe. A difficulty with this technique is that the resulting terminalrestriction fragments (TRFs) contain a 3-5 kbp stretch of subtelomericDNA that lacks restriction sites and thereby adds significantly to thesize of the measured telomere length. Another approach to eliminate thisDNA and improve accuracy of telomere length assays utilizes the factthat this subtelomeric DNA contains G and C residues in both strands,and thus should be cleaved under conditions that cause breaks at Gresidues In contrast, DNA composed exclusively of telomeric repeats willhave one strand lacking G residues, and this strand should remain intactunder G-cleavage conditions. The Maxam-Gilbert G-reaction usespiperidine to cleave guanine residues that have been methylated bydimethylsulfate (DMS) treatment. Although the original conditions of theMaxam-Gilbert G-reaction (treatment in 1M piperidine for 30 min. at 90°C.) breaks unmethylated DNA into fragments of 1-2 kbp and is thusnon-specific, milder conditions (0.1M piperidine for 30 min. at 37° C.)leave untreated DNA intact. The DNA is therefore treated with DMS andpiperidine as described above, precipitated with ethanol,electrophoresed, and hybridized on a Southern blot to the a (TTAGGG)₃probe. The results of such a test are shown in FIG. 26.

[0187] Telomerase Activity

[0188] Telomerase activity is useful as a marker of growth potential,particularly as to neoplastic cells, or progenitor cells, e.g.,embryonic stem cells. Human telomerase activity may be determined bymeasuring the rate of elongation of an appropriate repetitive sequence(primer), having 2 or more, usually 3 or more, repeats of the telomereunit sequence, TTAGGG. The sequence is labeled with a specific bindingpair member at a convenient site, e.g., the 5′-terminus, and thespecific binding pair member allows for separation of extendedsequences. By using one or more radioactive nucleoside triphosphates orother labeled nucleoside triphosphate, as described previously, one canmeasure the incorporated radioactivity as cpm per unit weight of DNA asa function of unit of time, as a measure of telomerase activity. Anyother detectable signal and label may also be used, e.g., fluorescein.

[0189] The activity may be measured with cytoplasmic extracts, nuclearextracts, lysed cells, whole cells, and the like. The particular samplewhich is employed and the manner of pretreatment will be primarily oneof convenience. The pretreatment will be carried out under conditionswhich avoids denaturation of the telomerase, so as to maintain thetelomerase activity. The primer sequence will be selected or labeled soas to allow it to be separated from any other DNA present in the sample.Thus, a haptenic label may be used to allow ready separation of theelongated sequence, which represents the telomerase activity of thesample. The nucleoside triphosphates which may be employed may includeat least one nucleoside triphosphate which is labeled. The label willusually be radiolabel, but other labels may also be present. The labelsmay include specific binding pair members, where the reciprocal membermay be labeled with fluorescers, enzymes, or other detectable label.Alternatively, the nucleoside triphosphates may be directly labeled withother labels, such as fluorescent labels.

[0190] The sequence elongation usually will be carried out at aconvenient temperature, generally from about 20° C. to 40° C., and for atime sufficient to allow for at least about 100 bp to be added on theaverage to the initial sequence, generally about 30-90 minutes. Afterthe incubation time to allow for the telomerase catalyzed elongation,the reaction may be terminated by any convenient means, such asdenaturation, e.g., heating, addition of an inhibitor, rapid removal ofthe sequence by means of the label, and washing, or the like. Theseparated DNA may then be washed to remove any non-specific binding DNA,followed by a measurement of the label by any conventional means.

[0191] The determination of telomerase activity may be used in a widevariety of ways. It can be used to determine whether a cell isimmortalized, e.g., when dealing with tissue associated with neoplasia.Thus, one can determine at the margins of a tumor, whether the cellshave telomerase activity and may be immortalized. The presence andactivity of the telomerase may also be associated with staging of canceror other diseases. Other diagnostic interests associated with telomeraseinclude measurement of activity as an assay for efficacy in treatmentregimens designated to inhibit the enzyme.

[0192] Other techniques for measuring telomerase activity can useantibodies specific for the telomerase protein, where one may determinethe amount of telomerase protein in a variety of ways. For example, onemay use polyclonal antisera bound to a surface of monoclonal antibodyfor a first epitope bound to a surface and labeled polyclonal antiseraor labeled monoclonal antibody to a second epitope dispersed in amedium, where one can detect the amount of label bound to the surface asa result of the telomerase or subunit thereof bridging between the twoantibodies. Alternatively, one may provide for primers to the telomeraseRNA and using reverse transcriptase and the polymerase chain reaction,determine the presence and amount of the telomerase RNA as indicative ofthe amount of telomerase present in the cells.

[0193] The following examples are offered by way of illustration and notby way of limitation.

EXAMPLES

[0194] The following are examples of specific aspects of the inventionto merely illustrate this invention to those in the art. These examplesare not limiting in the invention, but provide an indication of specificmethodology useful in practice of the invention. They also provide clearindication of the utility of the invention and of the correlationbetween telomere length, telomerase activity and cellular senescence.Such correlation indicates to those in the art the breadth of theinvention beyond these examples.

Example 1 Telomere Length and Cell Proliferation

[0195] The effects of telomere length modulation on cellularproliferation were studied. An average of 50 bp are lost per celldivision in somatic cells. The telomere end is thought to have asingle-stranded region as follows (although the amount of overhang isunknown):

[0196] 5′TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTAGGGTTAGGGTTAG GGTTA GGG3′AATCCCAATCCC (Seq. ID No. 1)

[0197] Applicant postulated that loss of this single-stranded overhangshould be significantly slowed if cells were provided with a syntheticoligonucleotide of the sequence CCCTAACCCTAA (Seq. ID No. 2). Thisoligonucleotide should hybridize to the exposed single-stranded region,and serve as a primer for DNA synthesis by the normal DNA polymerasepresent in somatic cells. In this way, rather than shortening by anaverage of 50 bp per division, the telomeres may only shorten by alesser amount per division, thus significantly extending the number ofdivisions required before telomere shortening induced cellularsenescence. This hypothesis was tested by measuring both the change inproliferative lifespan and rate of telomere shortening in cultured cellstreated with this indicated oligonucleotide, versus controloligonucleotides.

[0198] The efficacy of the CTO-12 oligonucleotide (5′-CCCTAACCCTAA-3′Seq. ID No. 2) to reduce telomere shortening associated with cellularsenescence (FIG. 1) was studied using target cells cultured understandard cell culture conditions in minimal essential mediumsupplemented with 10% fetal calf serum. The cells were subcultivatedevery four days by trypsinization upon reaching confluency and were fednew medium at subcultivation or every two days, whichever came first.Cells at various population doubling levels were seeded at 10,000 cellsper well and fed medium containing oligonucleotides at variousconcentrations. oligonucleotides studied were the cytidine-rich terminaloligonucleotide (CTO-12), guanidine-rich terminal oligonucleotide-12 bp(GTO-12, having the sequence 5′-TTAGGGTTAGGG-3′ (Seq. ID No. 3)), and a12 base pair randomer with a random nucleotide in every position. As anadditional control, cells were fed identical medium withoutoligonucleotide. Cells were fed oligonucleotide every 48 hours from 10Xstocks. (Such oligonucleotides may be modified to enhance stability,e.g., with phosphorothioates, dithioate and 2-O-methyl RNA.) In the caseof phosphorothioates it would be desirable to use longer CTO primerssuch as 5′-CCCTAACCCTAACCCT-3′, 5′-CCCTAACCCTAACCCTAA-3′, or5′-CCCTAACCCTAACCCTAACC-3′.

[0199] Specifically, IMR-90 human lung fibroblasts with a proliferativecapacity of approximately 55 population doubling (PD) were seeded atPD45 at 10,000 cells per well in a 48 well tissue culture dish, and fedmedium only or medium supplemented with CTO-12 (at 1.0 μM and 0.1 μM)and 12 base pair randomer at 1.0 μM. As shown in FIG. 1, cells grown inmedium without oligonucleotide, or with CTO-12 at less than 1.0 μM orwith oligonucleotide of random sequence reached replicative senescencein a similar fashion at about 52 population doubling. Cells fed theCTO-12 oligonucleotide at 1.0 μM, however, continued to proliferate forapproximately 10 doubling more than control cells.

Example 2 Inhibition of Telomerase in Cancer Cells

[0200] One way by which cancer cells are able to escape cellularsenescence is by regaining telomerase activity, which permits them tomaintain the length of their telomeres in the face of multiple rounds ofcell division. The enzyme telomerase contains an RNA complementary toTTAGGG, which allows it to recognize the telomeres and extend them bythe addition of additional TTAGGG repeats. In fact, one assay fortelomerase uses a TTAGGGTTAGGG primer and measures the ability of cellextracts to synthesis a ladder of 6 bp additions to this substrate.Telomerase activity in cancer cells is likely to be present in limitingamounts since telomere length is relatively stable (thus only about 50bp per telomere are added, so that lengthening and shortening arebalanced).

[0201] Applicant hypothesized that feeding cells a syntheticTTAGGGTTAGGG oligonucleotide (Seq. ID No. 3) should competitivelyinhibit the ability of telomerase to elongate chromosome ends, and thusshould lead to telomere shortening and senescence in cancer cells. Sincesomatic cells lack telomerase activity, the effects of this treatmentshould be strictly limited to cancer cells and the germ line.

[0202] Specifically, MDA 157 human breast cancer cells with an immortalphenotype were seeded at 10,000 cells per well in 12 well tissue culturedishes and fed medium only or medium supplemented with GTO-12 (at 1.0μM, 0.1 μM, and 0.01 μM). As shown in FIG. 2, cells grown in mediumwithout oligonucleotide, or with doses of less than 1.0 μM continuedreplicating in an immortal phenotype. Cells fed the CTO-12oligonucleotide, at 1.0 μM, however, ceased to proliferate after lessthan 10 doubling. Cells grown in the presence of 1.0 μM CTO-12 or 1.0 μMCTO-12 and 1.0 μM GTO-12 (G+C) continued to express the immortalphenotype suggesting that the GTO-12 oligonucleotide was notintrinsically toxic (FIG. 3). The lack of effect of the G+C mixture mayreflect the CTO-12 oligonucleotide, competing with or base pairing withthe GTO-12 oligonucleotide, this preventing its inhibitory effect on thecancer cell telomerase.

Example 3 Telomere Length as a Biomarker

[0203] In the U.S. and Western Europe, atherosclerosis is the principalcontributor to mortality from cardiovascular diseases (Ross, 314 N.Engl. J. Med. 488, 1986). Atherosclerosis is characterized by the muraland focal formation of lipid and cell-rich lesions or “plaques” on theintimal surfaces of arterial tissues. This is followed by anage-dependent expansion of the lesion into the lumen, potentiallyleading to occlusion and to myocardial and/or cerebral infarction(Haust, (1981) in Vascular Injury and Atherosclerosis, ed. Moore, S.(Marcel Dekker Inc., New York), pp. 1-22; Ross and Glomset, 295(7) N.Engl. J. Med. 369, 1976; and Ross, 295(8) N. Engl. J. Med. 420, 1976).Prominent among the mechanisms proposed to explain the pathogenesis ofatherosclerosis is the “response-to-injury” hypothesis (Ross, 314 N.Engl. J. Med. 488, 1986; Moore, (1981) in Vascular Injury andAtherosclerosis, ed. Moore, S. (Marcel Dekker Inc., New York), pp.131-148; and Moore, 29(5) Lab. Invest. 478, 1971) in which repeatedmechanical, hemodynamic and/or immunological injury to the endotheliumis the initiating event.

[0204] A prediction of this hypothesis is that the intimal and medialtissue in the area comprising the atherosclerotic plaque will have ahigher rate of cell turnover than the surrounding normal tissue. Severallines of evidence support this prediction. Ross et al., (Ross andGlomset, 295(7) N. Engl. J. Med. 369, 1976; Ross, 295(8) N. Engl. J.Med. 420, 1976) showed that cultured smooth muscle cells from fibrousplaques displayed lower responsiveness to growth serum when compared tocells from the underlying medial layer. Moss and Benditt 78(2) (1973)Am. J. Pathol. 175, 1973, showed that the replicative life-span of cellcultures from arterial plaques were equal to or less than thereplicative life-spans from cells of nonplaque areas. Dartsch et al., 10Arteriosclerosis 62, 1992, showed that human smooth muscle cellsobtained from primary stenosing lesions became senescent in culture farlater than smooth muscle cells from restenosing lesions. These resultssuggest that cells derived from regions of atherosclerotic plaquesundergo more cellular divisions than cells from non-plaque areas hencerendering them older and nearer to their maximum replicative capacity.

[0205] Thus, to understand the pathogenesis of atherosclerosis, one mustexamine the alterations in the behavior of cell turnover on and adjacentto the arterial lesions. One requires a biomarker for the cell turnoverof intimal and medial tissue. Several workers have examined hiomarkersfor the progression of atherosclerosis or for the propensity of anindividual to develop atherosclerosis. The former objective entailed themeasurement of a number of biochemical compounds which are detected inthe plasma but originate from the endothelium. Examples are serum TypeIII collagen (Bonnet et al., 18 Eur. J. Clin. Invest. 18, 1988), vonWillebrand's Factor (Factor VIII) (Baron et al., 10 Arteriosclerosis1074, 1990), cholesterol, triglycerides, apolipoprotein B (Stringer andKakkar, 4 (1990) Eur. J. Vasc. Surg. 513, 1990), lipoprotein (a)(Breckenridge, 143 Can. Med. Assoc. J. 115, 1990; Mezdour et al., 48Ann. Biol. Clin. (Paris) 139, 1990; and Scanu, 14 Clin. Cardiol. 135,1991), endothelin (Lerman et al., 325 N. Engl. J. Med. 997, 1991) andheparin-releasable Platelet Factor 4 (Sadayasu et al., 14 (1991) Clin.Cardiol. 725, 1991). A number of markers originate from the cell surface(Hanson et al., 11 (1991) Arterioscler. Thromb. 745, 1991; and Cybulskyand Girnbrone, 251 Science 788, 1991). Other markers monitorphysiological aberrations as a result of atherogenesis (Vita et al., 81(1990) Circulation 491, 1990). Candidate genes used to delineate theRFLP profile of those susceptible to atherogenesis (Sepehrnia et al., 38(1988) Hum. Hered. 136, 1988; and Chamberlain and Galton, 46 Br. Med.Bull. 917, 1990) have also been established. However, there have beenrelatively few markers developed to monitor directly cell turnover.

[0206] Applicant now shows that telomere length may serve as a biomarkerof cell turnover in tissues involved in atherogenesis. The results showthat endothelial cells lose telomeres in vitro as a function ofreplicative age and that in vivo telomere loss is generally greater fortissues of the atherosclerotic plaques compared to control tissue fromnon-plaque regions.

[0207] In general, telomere lengths were assessed by Southern analysisof terminal restriction fragments (TRF, generated through HinfI/RsaIdigestion of human genomic DNA. TRFs were resolved by gelelectrophoresis and hybridized with a telomeric oligonucleotide(³²P-(CCCTAA)₃) (Seq. ID No. 4). Mean TRF length decreased as a functionof population doubling in human endothelial cell cultures from umbilicalveins (m=−190 bp/PD, P=0.01), and as a function of donor age in iliacarteries (m=−120 bp/PD, P=0.05) and iliac veins (m=−160 bp/PD, P=0.05).Thus, mean TRF length decreased with the in vitro age of all cellcultures. When early passage cell cultures were assessed for mean TRFlength as a function of donor age, there was a significant decrease foriliac arteries (m=−102 bp/y, P=0.01) but not for iliac vein (m=47 bp/y,P=0.14). Mean TRF length of medial tissue decreased significantly(P=0.05) as a function of donor age. Intimal tissues from one individualwho displayed extensive development of atherosclerotic plaques possessedmean TRF lengths close to those observed for senescent cells in vitro(−6 kbp). These observations indicate that telomere size indeed servesas a biomarker for the replicative history of intima and media and thatreplicative senescence of endothelial cells is involved inatherogenesis.

[0208] Specifically, the following materials and methods were used toachieve the results noted below.

[0209] Endothelial Cell Cultures Human umbilical vein endothelial cells(HUVEC) were obtained from Dr. Thomas Maciag of the Jerome H. HollandLaboratory of the American Red Cross. Human endothelial cells from theiliac arteries and iliac veins were obtained from the Cell Repository ofthe National Institute of Aging (Camden, N.J.). Cells were grown at 37°C. in 5% CO₂ on 100 mm tissue plates whose interiors were treated withan overnight coating of 0.4% gelatin (37° C.). The supplemented mediaconsisted of M199, 15% fetal bovine serum, 5 U/ml heparin and 20 μg/mlcrude Endothelial Cell Growth Supplement (Collaborative Research) orcrude Endothelial Cell Growth Factor (Boehringer-Mannheim). Cultureswere trypsinized (0.05%, 3 minutes) at confluence, reseeded at 25% ofthe final cell density and refed every 2-3 days.

[0210] Tissue Samples Tissue samples from the aortic arch, abdominalaorta, iliac artery and iliac vein were obtained from autopsies at theDepartment of Pathology, Health Sciences Center, McMaster University.Post-mortem times ranged from 5 to 8 hours. The intima was obtained bycutting open the arteries or veins and carefully scraping off thelumenal surface with a No. 10 scalpel (Lance Blades, Sheffield) (Ryan,56 Envir. Health Per. 103, 1984). The resulting material was eithertreated directly for extraction of DNA or processed for cell culture.

[0211] The adventitial layer was removed by cutting or scraping thenon-lumenal side of the vessel. The remaining medial layer was preparedfor DNA extraction by freezing it in liquid-N₂ and grinding it in aliquid-N₂ chilled mortar and pestle (Kennedy et al., 158 Exp. Cell Res.445, 1985). After the tissue was ground to a powder, 5 ml of frozendigestion Buffer (10 mM Tris; 100 mM NaCl; 25 mM EDTA; 0.5% SDS; pH 8.0)was added and ground into the powderized tissue. The powder was thentransferred to a 50 ml Falcon tube and incubated at 48° C. until thawed.Proteinase K (10 mg/ml) was added to a final concentration of 0.2 mg/ml.After a 12-16 hour incubation, the solution was removed from the waterbath and either prepared for DNA extraction or stored at 20° C.

[0212] Extraction and Restriction Enzyme Digestion of Genomic DNA

[0213] DNA was extracted as described previously (Harley et al., 345Nature 458, 1990; Allsopp et al., 89 Proc. Natl. Acad. Sci. USA 10114,1992). In brief, proteinase K-digested lysates were extracted twice withone volume of phenol:chloroform:isoamyl alcohol (25:24:1) and once withchloroform. Nucleic acid was precipitated by adding 2 volumes of 100%EtOH to the aqueous layer, washed once with 70% EtOH and finallyresuspended in 100-200 μl of 10 mM Tris-HCl, 1 mM EDTA, pH 7.5. DNA wasquantified by fluorometry and 1 μg was digested with 1 unit each ofHinfI/RsaI for 3-24 hours at 37° C. Complete digestion was monitored bygel electrophoresis. The integrity of the DNA before and after digestionwas monitored in control experiments by gel electrophoresis.

[0214] Southern Blot Hybridization

[0215] Electrophoresis of digested genomic DNA was performed in 0.5%agarose gels in a standard Tris, sodium borate, EDTA buffer for a totalof 650-700 V/hr as described previously (Harley et al., 345 Nature 458,1990; Allsopp et al., 89 Proc. Natl. Acad. Sci. USA 10114, 1992). Afterelectrophoresis, the gel was placed onto 3 mm Whatman filter paper anddried under vacuum for 25 minutes at 60° C. Gels were denatured bysoaking in 0.5 M NaOH, 1.5 M NaCl for 10 minutes at room temperature andthen neutralized through immersion in 0.5 M Tris, 1.5 M NaCl. GenomicDNA was immersed in standard hybridization solution (Harley et al., 345Nature 458, 1990) (6×SSC) with the telomeric ³²P-(CCCTAA)₃ probe (Seq.ID No. 4) for 12-16 hours at 37° C. The telomeric smears were visualizedthrough autoradiography on pre-flashed (OD₅₄₅=0.15) Kodak XAR-5 film.The mean lengths of the terminal restriction fragments (TRFs) werecalculated from densitometric scans of the developed films as describedpreviously (Harley et al., 345 Nature 458, 1990).

[0216] In vitro Results

[0217] To determine the feasibility of employing telomere length as abiomarker for cell turnover in atherosclerosis, we first examined thechange in telomere length in cultured endothelial cells where celldivision can be directly monitored in vitro. The DNA was digested withHinfI and RsaI, and the resulting terminal restriction fragments (TRF)were subjected to Southern analysis. As in human skin fibroblasts(Allsopp et al., 89 Proc. Natl. Acad. Sci. USA 10114, 1992), mean TRFlength decreased as a function of population doubling (PD). Thus,telomere length decreases with in vitro age of human umbilical veinendothelial cells. Mean TRF length decreased linearly (P=0.01) at a rateof 190±10 bp/PD (see FIG. 4). The Y-intercept, which signifies the meanTRF at 0 PDL is 14.0 kbp while mean TRF at senescence was 5.7±0.4 kbp.

[0218] To prove that telomere length decrease occurred in endothelialcells from other arterial and venous sources, mean TRF length versuspopulation doubling level (PDL) was determined for several strains ofendothelial cells from human iliac artery and human iliac vein. In bothiliac arteries and iliac veins there was a significant (P=0.05) lineardecrease in mean TRF length with age of culture: 120±60 bp perpopulation doubling for the iliac artery and 160±30 bp per populationdoubling for the iliac veins from endothelial cells.

[0219] In vivo Results

[0220] Formation of atherosclerotic plaques occurs more often in theiliac artery than in the iliac vein (Crawford, (1982) Pathology ofAtherosclerosis (Butterworth and Co. Ltd., U.K.), p. 187-199), thus itis expected that turnover of intimal tissue in vivo from the iliacartery should be greater than that from the iliac veins. To test this,nine different strains of endothelial cell cultures from iliac arteriesand veins of donors ranging in age from 14-58 years of age werecultivated and TRF lengths from the earliest possible PDL weredetermined (FIG. 5).

[0221] Consistent with the hypothesis of greater cell turnover in vivoin arteries than in veins, the rate of decrease in mean TRF length, wassignificant over the age range 20-60 years for iliac arteries (−100bp/yr, P=0.01) and greater than for the iliac veins (−47 bp/yr, P=0.14).Among the nine strains of endothelial cells, there were cultures fromthe iliac artery and iliac vein from the same individuals for 3 of thedonors, aged 21, 47 and 49 years. There was a significantly shorter meanTRF length in the cultures of iliac artery cells as compared to thevenous cells for the two older donors. The younger donor showed nosignificant difference in mean TRF length between the two cultures,possibly reflecting relatively little difference in cell turnoverbetween the vessels of the 21-year old donor.

[0222] Differences in mean TRF length of the cell cultures from iliacarteries and iliac veins in donors of different ages will reflect notonly differences in original mean TRF length of the primary tissues butalso differences in the rate of telomere loss between the differentcultures in vitro during the time required to collect sufficient cellsfor analysis (approximately 5-10 PDL). To determine if there is arelationship between cell turnover and the extent of atheroscleroticplaque formation, we examined mean TRF length in primary tissue.Autopsies from 3, 11, 12, 14, 18, 26, 75-year old females and a 77-yearold male were performed. Sections of the aortic arch, abdominal aorta,iliac artery and iliac vein were taken and the intimal and medialtissues separated and assessed for TRF length.

[0223] Sufficient intimal tissue could be obtained from the aortic arch,abdominal aorta, iliac arteries and iliac veins of 3 donors (aged 27, 75and 77 years) for TRF analysis. There was a striking difference betweenthe mean TRF lengths averaged over these sites in the 27-year old female(10.4±0.7 kbp) versus the 75-year old (8.8±0.6 kbp) and the 77-year oldmale (6.3+0.4kbp). It is noteworthy that the 77-year old male hadextensive atherosclerotic lesions in his vasculature and that the meanTRF length of his intimal tissue is close to that of endothelial cells,at senescence in vitro (approximately 6 kbp, FIG. 4).

[0224]FIG. 6 shows that mean TRF of medial tissue (from the aortic arch)decreases with donor age at a small but significant rate (47 bp/yr,P=0.05). Thus, medial cells turnover in vivo occurs at a rate less thanthat of the venous or arterial endothelial cells.

[0225] In general, telomere loss in medial tissue underlying anatherosclerotic plaque was greater than those in non-plaque regions(Table 1). With the 75-year old female, mean TRF was significantlyreduced in medial DNA from the plaque regions versus the non-plaqueregions of both the aortic arch (P=0.04) and the abdominal aorta(P=0.01). For the 77-year old male, this was observed in the abdominalaorta (P=0.01). TABLE 1 Mean TRF values for primary medial tissues ofplaque and non-plaque areas Plaque Region Non-Plaque Region P 75-yearold Donor Aortic Arch 10.2 ± 0.5  11.1 ± 0.1 0.04 Abdominal Aorta 9.5 ±0.6 11.0 ± 0.1 0.01 77-year old Donor Aortic Arch 8.2 ± 0.4  8.4 ± 0.2NS Abdominal Aorta 7.1 ± 0.1  8.2 ± 0.4 0.01

[0226] These results show that mean TRF length decreases as a functionof donor age for primary medial and intimal tissue, suggesting that cellturnover does occur in cardiovascular tissue. The decrease in mean TRFlength for plaque regions versus clear regions of medial tissue from thesame blood vessel is consistent with augmented cell turnover of tissueassociated with atherosclerotic plaques. Thus, the results indicate thatmeasurement of telomere length provides a biomarker for alterations ofcellular turnover in tissues associated with cardiovascular diseases,i.e., cells of the intima and media.

[0227] Measurement of telomere length is a direct register ofproliferative history but to obtain telomeric DNA one must obtain abiopsy of endothelial tissue. Since removal of the endothelium in itselfcan induce plaque formation, the biopsy strategy obviously entailsethical and practical problems. Based upon experience with autopsysamples one requires a minimal area of 1 cm² in order to perform aSouthern analysis as described in this paper. For a practical biopsy,this is untenable. A detection technique to circumvent this problem maybe confocal fluorescent microscopy.

Example 4 Simplified Test for Telomere Length

[0228] Telomere length has been found to be the best predictor of theremaining lifespan of cells cultured from donors of different ages. Theability to measure telomere length thus has significant clinical use.Because of their simple repetitive nature, telomeres lack DNA sequencesrecognized by many restriction enzymes. One way to measure telomerelength is to digest DNA with restriction enzymes with 4-base recognitionsites, which cuts most of the DNA into very small pieces and leaves thetelomeres in relative large TRFs (Terminal Restriction Fragments). ASouthern blot of the DNA is then probed with a radioactiveTTAGGGTTAGGGTTAGGG (Seq. ID No. 5) oligonucleotide, and the size of theTRF determined.

[0229] A much simpler method to measure telomere length exploits thefact that the telomere sequence lacks guanidine residues in the C-richstrand. Genomic DNA can be melted and mixed with the DNA synthesisprimer CCCTAACCCTAACCCTAACCCTAA (Seq. ID No. 6) in the presence of DNApolymerase and only three deoxynucleotides (DATP, dTTP and radioactivedCTP). Rare complementary sequences scattered throughout the genomewould fail to extend due to the lack of dGTP. The length of the extendedDNA can then be determined from a simple gel electrophoresis. The amountof DNA synthesized (counts incorporated per μg of DNA) will be directlyproportional to telomere length, and for diagnostic purposes a simplemeasure of radioactivity would then suffice to quantitate telomerelength.

Example 5 Identification of DNA Sequences Near Telomeres

[0230] There are good reasons to believe that the regulatory factorsthat control cellular and organismal senescence are located neartelomeres, and are themselves regulated by the length of the adjacenttelomere. It is thus important to identify and clone them in order to beable to understand and manipulate the aging process. In addition, thereis great interest in identifying unique telomeric DNA within the humangenome project, since telomeric markers for mapping purposes are lackingfor the ends of the chromosomes.

[0231] In one method, large telomeric DNA is purified as follows. Abiotinylated CCCTAACCCTAA (Seq. ID No. 7) oligonucleotide is used toprime DNA synthesis in double-stranded genomic DNA. The only sequenceswith which this oligonucleotide can anneal will be the single-strandedbase overhangs at telomere ends. The extended DNA will then be digestedwith a restriction enzyme such as NotI to produce large restrictionfragments. Biotinylated fragments are retrieved using streptavidincoated magnetic beads, and analyzed by pulsed field electrophoresis. 46fragments (one for each end of the 23 human chromosomes) are produced.

[0232] Multiple strategies can be used to pursue the successfulisolation of large telomeric DNA. The DNA can be labeled and used toscreen cDNA libraries in order to identify genes located near telomeres.The expression of these cDNAs can then be examined in young versus oldcells in order to identify those which are differentially expressed as afunction of cellular senescence, and which are thus candidates to beregulatory factors that control aging.

[0233] The purified telomeric DNA can also be digested with additionalrestriction enzymes, mixed with 100-fold excess of genomic DNA, meltedand reannealed. Under these circumstances, the repetitive sequences inthe telomeric DNA will anneal with genomic DNA while unique sequences inthe purified DNA will self-anneal. Only the self-annealed uniquesequences will contain restriction overhangs at each end, and thus asimple cloning of the annealed DNA will result in the successful cloningof only unique fragments.

Example 6 Telomere Loss in Down's Syndrome Patients

[0234] Loss of telomeric DNA from human chromosomes may ultimately causecell cycle exit during replicative senescence. Since lymphocytes have alimited replicative capacity and blood cells were previously shown tolose telomeric DNA during aging in vivo, we wished to determine whetheraccelerated telomere loss is associated with the prematureimmunosenescence of lymphocytes in individuals with Down's Syndrome(DS), and whether telomeric DNA is also lost during aging of lymphocytesin vitro.

[0235] To investigate the effects of aging and trisomy 21 on telomereloss in vivo, genomic DNA was isolated from peripheral blood lymphocytesof 140 individuals (0-107 y) and 21 DS patients (0-45 y). Digestion withrestriction enzymes HinfI and RsaI generated terminal restrictionfragments (TRFs) which can be detected by Southern analysis using atelomere-specific probe, (³²P-(CCCTAA)₃). The rate of telomere loss wascalculated from the decrease in mean TRF length as a function of donorage. DS patients showed a significantly higher rate of telomere losswith donor age (133±15 bp/y) compared to age-matched controls (41±7.7bp/y) (P<0.0005), indicating that accelerated telomere loss is abiomarker of premature immunosenescence of DS patients, and may play arole in this process.

[0236] Telomere loss during aging in vitro was calculated forlymphocytes from two normal individuals grown in culture for 20-30population doubling. The rate of telomere loss was 90 bp/cell doubling,that is, it was comparable to that seen in other somatic cells. Telomerelengths of lymphocytes from centenarians and from older DS patients weresimilar to those of senescent lymphocytes in culture, which suggeststhat replicative senescence could partially account for aging of theimmune system in DS patients and elderly individuals.

[0237] The following materials and methods were used to obtain theresults provided below.

[0238] Culture of Human Peripheral Blood T Lymphocytes

[0239] Adult peripheral blood samples were collected, and mononuclearcells were isolated by Ficoll-Hypaque gradient centrifugation thencryopreserved in liquid nitrogen. Cultures were initiated by mixing 10⁻⁶mononuclear cells with 10⁶ irradiated (8000 Rad) lymphoblastoid cells(Epstein-Barr virus transformed B cells), or 10⁶ mononuclear cells with10 μg/ml phytohemagglutinin (PHA-P, Difco) in each well of a 48-wellcluster plate (Costar). After 8 to 11 days, cells were washed and platedin 2 ml wells of 24-well cluster plates at a concentration of2-4×10⁵/ml. Cultures were passaged every three to four days, or wheneverviable cell concentration (determined by trypan blue exclusion) reached≧8×10⁵/ml. Cultures were terminated when they showed no proliferativeresponse to irradiated lymphoblastoid cells and/or when there were noviable cells present in the entire visual field of the haemocytometer.Once transferred to the 2 ml wells, cells were continuously exposed to25 U/ml of recombinant interleukin-2 (Amgen). The media used were (a)RPM1 (Irvine Scientific) supplemented with 10 to 20% fetal calf serum, 2mM glutamine, and 1 mM Hepes; (b) AIM V™, a DMEM/nutrient mixture F-12basal medium, containing purified human albumin, transferrin, andrecombinant insulin (Gibco), supplemented with 25% Ex-cyte (an aqueousmixture of lipoprotein, cholesterol, phospholipids, and fatty acids,(Miles Diagnostics).

[0240] At each cell passage, the number of population doubling (PD) wascalculated according to the formula: PD=ln (final viable cell no.initial cell no.)/ln2.

[0241] Isolation of DNA

[0242] PBLs (including=15% monocytes) were isolated using Ficoll-Hypaquegradient centrifugation (Boyum et al., 21(97) Scan. J. Clin. Lab.Invest. 77, 1968) and washed 3 times in PBS. Cell pellets wereresuspended in 500 μl of proteinase K digestion buffer (100 mM NaCl, 10mM Tris pH 8, 5 mM EDTA, 0.5% SDS) containing 0.1 mg/ml proteinase K andincubated at 48° C. overnight. Lysates were extracted twice withphenol/chloroformisoamyl alcohol (25:24:1 v/v/v) and once withchloroform. DNA was precipitated with 95% ethanol and dissolved in TE(10 mM Tris, 1 mM EDTA, pH=8).

[0243] Analysis of Telomeric DNA

[0244] Genomic DNA (10 μg) was digested with HinfI and RsaI (BRL) (20 Ueach), re-extracted as above, precipitated with 95% ethanol, washed with70% ethanol, dissolved in 50 μl TE, and quantified by fluorometry. Oneμg of digested DNA was resolved by electrophoresis in 0.5% (w/v) agarosegels poured on Gel Bound (FMC Bioproducts) for 700 V-h. Gels were driedat 60° C. for 30 minutes, denatured, neutralized, and probed with5′end-labeled ³²P-(CCCTAA) as described above. Autoradiograms exposedwithin the linear range of signal response were scanned with a Hoeferdensitometer. The signal was digitized and subdivided into 1 kbpintervals from 2 kbp to 21 kbp for calculation of the mean TRF length(L) using the formula L=Σ, (OD_(i−)L_(i))/ΣOD_(i), whereOD_(i)=integrated signal in interval i, and L=TRF length at themid-point of interval i.

[0245] TRF Length vs. Age

[0246] When measured as a function of donor age, mean TRF length in PBSof 140 unrelated normal individuals (aged 0-107 y) declined at a rate of41±2.6 bp/y (p<0.00005, r=0.83). This rate of TRF loss for PBLs is closeto that previously found for peripheral blood cells by Hastie et al.,346 Nature 866, 1990. When our data were separated according to genderit was noticed that males lost telomeric DNA at a rate slightly fasterthan that of females (50±4.2 vs 40±3.6 bp/y), but this difference didnot reach statistical significance (p=0.1). The 18 centenarians (aged99-107 y) among our population of normal individuals had a mean TRFlength of 5.28±0.4 kbp (FIG. 7). Interestingly, the standard deviationof mean TRF values for the centenarians (0.4 kbp) was much smaller thanthat of other age groups. Although it is possible that this representsselection of a more homogeneous population of cells with age, it is alsopossible that the group of centenarians were less genetically diversethan the younger populations in our study.

[0247] Mean TRF length was also analyzed in PBLs of 21 Down's Syndromeindividuals (aged 2-45 y) and the rate of loss was compared to 68age-matched controls (aged 0-43 y). We found that cells from DS patientsshowed a significantly greater rate of telomere loss (133±15 bp/y vs41±7.7 bp/y; one tailed t-test, t=5.71, p<0.0005) (FIG. 8).

[0248] To determine the rate of telomere loss as a function of celldoubling, we cultured normal lymphocytes from 2 individuals in vitrountil replicative senescence and measured mean TRF length at severalpopulation doubling levels (FIG. 9). Mean TRF length decreased 90bp/population doubling in these strains, within the range observed forother human somatic cell types. The mean TRF length at senescence forthe lymphocyte cell strains shown here and one other analyzed atterminal passage (FIG. 9), was 5.1±0.35 kbp. The observed TRF values invivo for PBLs of centenarians (5.3±0.4 kbp) and old DS patients(4.89±0.59 kbp), were close to this value, suggesting that a fraction ofthe cells from these individuals were close to the limit of theirreplicative capacity.

[0249] The results showing that telomeres in PBLs from normalindividuals shorten during aging in vivo and in vitro extend similarobservations on human fibroblasts (Harley et al., 345 Nature 458, 1990)and support the hypothesis that telomere loss is involved in replicativesenescence. We also found that in Down's Syndrome, the rate of telomereloss in PBS in vivo was significantly higher than that in age-matchednormal donors. Thus, accelerated telomere loss in PBS of trisomy 21, asyndrome characterized by premature immunosenescence and other featuresof accelerated aging (Martin, “Genetic Syndromes in Man with PotentialRelevance to the Pathobiology of Aging”, in: Genetic Effects on Aging,Bergsma, D. and Harrison D. E. (eds.), pp. 5-39, Birth Defects: Originalarticle series, no. 14, New York: Alan R. Liss (1978)), could reflectearly senescence of lymphocytes.

[0250] The increased rate of telomere loss in PBS from DS patients couldreflect a higher turnover rate of cells in vivo due to reduced viabilityof the trisomy 21 cells. However, it is also possible that the rate oftelomere loss in PBS from DS patients is greater per cell doubling thanthat in normal individuals.

[0251] The pathology of DS is similar in many ways to normal aging.Premature senescence of the immune system possibly plays a role in thissimilarity since DS patients have a high incidence of cancer and sufferfrom autoimmunity. In support of this idea, lymphocytes of older DSpatients and old individuals share several characteristics, includingdiminished response of T-cells to activate and proliferate in responseto antigen, low replicative capacity, and reduced B- and T-cell counts(Franceschi et al., 621 Ann. NY Acad. Sci. 428, 1991). Our finding thattelomere length decreased faster in DS patients than normal individuals,and that the mean TRF length in centenarians and old DS patients in vivowere similar to that of senescent lymphocytes in vitro (=5 kbp)1 extendsthese observations. Moreover, these data suggest that replicativesenescence within the lymphoid lineage in vivo contributes to thecompromised immune system of both elderly individuals and Down'sSyndrome patients.

Example 7 Ovarian Cancer and Telomerase Activity

[0252] The following is an example of a method by which telomeraseactivity is shown to correlate with the presence of cancer cells. Inaddition, the length of TRF was determined as an indication of thepresence of tumor cells. Generally, it was found that tumor cells hadsignificantly lower TRF values than surrounding normal cells, and hadtelomerase activity. Thus, these two features are markers for thepresence of tumor cells.

[0253] The following methods were used to obtain these results:

[0254] Separation of Tumor and Non-tumor Cells

[0255] In one method, ascitic fluid was obtained by either diagnosticlaparotomy or therapeutic paracentesis (from patients diagnosed ashaving ovarian carcinoma), and centrifuged at 600×g for 10 minutes at 4°C. The cell pellet was washed twice in 10 to 30 ml of phosphate bufferedsaline (PBS: 2.7 MM KCl, 1.5 mM KH₂PO₄, 137 mM NaCl and 8 mM Na₂HPO₄)and centrifuged at 570×g for 4 minutes at 4° C. After the final wash thecell pellet was resuspended in 20 ml of PBS and filtered through a 30 or10 μm nylon mesh filter (Spectrum) which retains the tumor clumps butnot single cells. The filters were backwashed to liberate highlypurified tumor clumps. The flow-through was a combination offibroblasts, lymphocytes and tumor cells.

[0256] In another method ascitic fluid cells were collected and washedas described above. The cellular pellet was resuspended in a-MEM with10% fetal calf serum and cultured in 150 mm dishes. After 12 hours themedia was removed and new plates were used to separate the adheringfibroblasts from the non-adhering cells in the medium. After 12 hoursthe media containing mostly tumor clumps was removed from the secondplates and allowed to adhere in DMA F12 medium supplemented with 3%fetal calf serum, 5 ng/ml EGF, 5 μg/ml insulin, 10 μg/ml humantransferrin, 5×10⁻⁵ M phosphoethanolamine and 5×10⁻⁵ M ethanolamine.These tumor cells were cultured for DNA analysis and S100 extracts.

[0257] DNA Extraction

[0258] Cells were lysed and proteins were digested in 10 mM Tris-Hcl (pH8.0), 100 mM NaCl, 25 mM EDTA, 0.5% SDS, 0.1 mg/ml proteinase K at 48°C. overnight. Following 2 extractions with phenol and 1 with chloroform,DNA was precipitated with ethanol and dissolved in 10 mM Tris-HCl (pH8.0), 1 mM EDTA (TE).

[0259] Determination of TRF Length and Amount of Telomeric DNA

[0260] Genomic DNA was digested with HinfI and RsaI, extracted andprecipitated as above, and redissolved in TE. DNA concentration wasmeasured by fluorometry (Morgan et al., 7 Nucleic Acids Res. 547, 1979).DNA samples (1 μg each) were loaded onto a 0.5% agarose gel andelectrophoresed for 13 hours at 90 V. The gel was dried at 60° C. for 30minutes, denatured in 1.5 M NaCl and 0.5 M NaOH for 15 minutes,neutralized in 1.5 M NaCl, 0.5 M Tris-HCl (pH 8.0) for 10 minutes andhybridized to a 5′ ³²P(CCCTAA)₃ telomeric probe in 5×SSC (750 mM NaCland 75 mM sodium citrate), 5×Denhart's solution (Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor (1982)) and 0.1×P wash (0.5 mM pyrophosphate, 10 mMNa₂HPO₄) at 37° C. for 12 hours. Following three high stringency washesin 0.24×SSC at 20-22° C. (7 minutes each), the gel was autoradiographedon pre-flashed (OD=0.15) Kodak XAR-5 X-ray films for 3 days withenhancing screens. Each lane was scanned with a densitometer and thedata used to determine the amount of telomeric DNA and the mean TRFlength as previously described (Harley et al., 345 Nature 458, 1990).

[0261] Preparations of S-100 Cell Extracts

[0262] A minimum of 6×10⁸ cells were used for each extract. Asciticfluid or purified ascitic fluid tumor cells (by the first methoddescribed above) were centrifuged at 570×g for 4 minutes at 4° C.Ascitic fluid tumor cells separated by the second method described above(grown in monolayer) were harvested by scraping with a rubber policeman,and centrifuged as above. The pellets were rinsed twice in cold PBSfollowed by centrifugation as above. The final pellet was rinsed in cold2.3×Hypo buffer (1×Hypo buffer: 10 mM Hepes (pH 8.0)), 3 mM KCl, 1 mMMgCl₂, 1 mM DTT, 0.1 mM PMSF and 10 U/ml of RNAsin, 1 μM leupeptin and10 μM pepstatin A, centrifuged for 5 minutes and resuspended in 0.75volumes of 2.3×Hypo buffer. After incubation on ice for 10 minutes thesample was transferred to an ice cold 7 or 1 ml Dounce homogenizer andhomogenized on ice using a B pestle (25-55 μm clearance). After afurther 30 minutes on ice the samples having a volume larger than 1 mlwere centrifuged for 10 minutes at 10,000 rpm (16,000×g) at 4° C. in aBeckman J3-13.1 swinging bucket rotor. One-fiftieth volume of 5 M NaClwas added, and the samples supernatant were centrifuged for 1 hour at38,000 rpm (100,000×g) at 4° C. in a Beckman Ti50 rotor. Glycerol wasadded to a final concentration of 20% and the extract aliquoted andstored at −70° C. Samples less than 1 ml were centrifuged at 55,000 rpmfor 1 hour at 4° C. in a TLA 100.2 rotor (Beckman) and NaCl and glycerolwere added to the supernatant as above. Protein concentration in atypical extract was approximately 4 mg/ml.

[0263] Telomerase Assay

[0264] Telomerase activity was assayed by a modification of the methodof Morin, 59 Cell 521, 1989. Aliquots (20 μl) of S-100 cell extract werediluted to a final volume of 40 μl containing 2 mM dATP, 2 mM dTTP, 1 mMMgCl₂, 1 μM (TTAGGG)₃ primer, 3.13 μM (50 μCi) a-³²P-dGTP (400Ci/mmole), 1 mM spermidine, 5 mM β-mercaptoethanol, 50 mM potassiumacetate, and 50 mM Tris-acetate (pH 8.5). In some experiments reactionvolumes were doubled. The reactions were incubated for 60 minutes at 30°C. and stopped by addition of 50 μl of 20 mM EDTA and 10 mM Tris-HCl (pH7.5) containing 0.1 mg/ml RNAseA, followed by incubation for 15 minutesat 37° C. To eliminate proteins, 50 μl of 0.3 mg/ml Proteinase K in 10mM Tris-HCl (pH 7.5), 0.5% SDS was added for 10 minutes at 37° C.Following extraction with phenol and chloroform, unincorporateda-³²P-dGTP was separated by centrifuging the samples for 4 minutes at500 g in a swinging bucket rotor through NICK SPIN columns (Pharmacia).DNA was precipitated by the addition of 5.3 μl of 4 M NaCl, 4 μg ofcarrier tRNA and 500 μl of ethanol at −20° C. DNA pellets wereresuspended in 3 μl of formamide loading dye, boiled for 1 minute,chilled on ice and loaded onto an 8% polyacrylamide, 7 M urea sequencinggel and run at 1700 V for 2 hours using 0.6×TBE buffer. Dried gels wereexposed to Kodak XAR-5 pre-flashed film at −70° C. with enhancing screenor to phosphoimager screens (Molecular Dynamics) for 7 days.

[0265] The results of the above experiments are shown in tables 2 and 3below: TABLE 2 Characteristics of ATCC Ovarian Carcinoma Cell Lines Cellline Mean TRF Length (kbp) Telomerase Activity HEY stable at 3.7 +CAOV-3 stable at 3.7 N.D. SKOV-3 Increases at 60 bp/pd N.D.

[0266] TABLE 3 Characteristics of Ovarian Carcinoma Tumor Cells fromAscitic Fluid Mean TPF Telomerase Patient Description Length (kbp)Activity Pres-3 Purified tumor cells 3.7 + Mac-2 Purified tumor cells3.7 N.D. Sib-1 Purified tumor cells 4.2 N.D. Ric 207 Purified tumorcells 3.3 N.D. Cra-1 Purified tumor cells 5.2 N.D. Ing-1 Purified tumorcells 5.8 N.D. Lep-1 Purified tumor cells 5.8 N.D. Lep-4 Purified tumorcells 5.6 N.D. Sal-1 Purified tumor cells 5.6 N.D. Rud-1 Ascitic fluidcells 3.4 + Murr-1 Ascitic fluid cells 3.8 + Dem-1 Ascitic fluid cellsN.D. + Cas-1 Ascitic fluid cells 5.3 + Wad-1,2 Ascitic fluid cells 4.9N.D.*

[0267] Table 4 shows the TRF length of cells from ascitic fluid. Aminimum of 2 autoradiographs were scanned with a densitometer over thesize range 2-21 kbp, and the densitometric values used to determine meanTRF length in kbp. Average standard deviation of the data was 0.5 kbpwith the largest deviation being 2 kbp. The value following the threecharacter patient code refers to the paracentesis number (i.e., OCl-1 isthe first sample from patient OCI). Samples defined as E (early) wereobtained near the time of presentation while samples L (late) wereobtained near death. Paracenteses were performed 4 to 15 times over thecourse of 4 to 22 months. TABLE 4 Unfractionated Fractionated Culturedcells TRF Normal Tumour Tumour* Patient (kbp) Patient TRF (kbp) TRF(kbp) Patient TRF (kbp) OC1-1 3.8 OC5-1  7.0 5.0 OC18-2 3.4 OC2-1 5.5OC6-1  9.2 5.4 OC19-3 3.4 OC3-1 5.4 OC7-1  8.0 5.4 OC20-1 4.2 -2 4.4OC8-1  7.7 4.3 OC21-1 3.3 OC4-1 4.5 OC9-1  5.2 OC5-1 4.3 OC10-1 3.9OC22-13 6.9 OC11-2 3.7 OC12-1 3.8 OC13-1 5.1 Serial Samples OC14-1 (E)9.4 5.0 -4 (L) 9.3 5.2 OC15-1 (E) 7.3 4.1 -5 (L) 4.7 OC16-1 (E) 3.9 -2(E) 3.4 -7 (L) 3.9 OC17-1 (E) 7.7 4.3 -15 (L) 4.7 Means⁵⁵⁴: 4.7 ± 0.78.2 ± 0.9 4.5 ± 0.8 3.7 ± 0.5 (4.2 ± 1.4)^(‡)

[0268] Table 5 shows the telomerase activity in normal and tumor cells.Leukocytes and acsites cells were isolated and ascitic fluid cellsfractionated into normal and tumor fractions and assayed for telomeraseactivity. Protein concentration in all extracts was <2 mg/ml, i.e., 20fold higher than the lowest concentration at which activity was detectedin control 293 CSH extract. TABLE 5 Unfractionated FractionatedTelomerase Telomerase activity Patient activity Patient Normal TumourOC4-1 + OC19-3 N.D. +   -5 + OC17-1 — N.D. OC2-1 ± OC8-1 — N.D. OC1-1 +LEK — N.D. OC23-1 +

[0269] In the TRF assay, each tumor clump had significantly lower TRFlengths than associated normal cells. (See FIG. 10).

[0270] In the telomerase assay, significantly greater telomeraseactivity was evident in the ascitic fluid of certain patients than inthe control tumor lines HEY and PRES, or the control cell line 293 CSH(FIGS. 11, 33).

Example 8 Effect of HIV Infection on TRF Length

[0271] HIV infection leads to an acute viral infection manifestingitself as a virus-like syndrome, followed by a prolonged period oflatency characterized by an absence of signs and symptoms. During thisprolonged asymptomatic period (lasting usually 7-10 years), there is nodiagnostic available for staging the course of the infection other thanthe presence or absence of antibodies to viral coat proteins. This doeslittle to stage the disease or to help the physician measure theeffectiveness of prophylactic agents.

[0272] While Meyaard et al., 257 Science 217, 1992, propose a programmedcell death for CD4⁺ and CD8⁺ cells of an HIV-infected individual, wepropose that during those 7 to 10 years the immune system is able tokeep the infection relatively repressed, but there is markedly increasedturnover of the infected CD4⁺ T-destruction. We propose that thisessentially accelerates the replicative senescence of this particularsubpopulation of T-cells, and with time results in a population ofprecursor pluripotent cells with markedly reduced proliferativecapacity. Finally, this results in CD4⁺ T-cells that are relativelyunresponsive to stimuli to proliferate, as is typical of the replicativesenescence of the cells observed in vitro.

[0273] We also propose that the replicative capacity of total peripherallymphocytes or CD4⁺ cells in particular, can be effectively determinedby assaying telomere repeat length utilizing the method described above,e.g., with the oligonucleotide probe 5′ TTAGGGTTAGGGTTAGGGTTAGGG (or oneof similar or complementary sequence) hybridized to CD4⁺ lymphocyte DNAisolated from the patient along with molecular size markers. Theseassays allow the physician to chart the course of the disease during thelong intervening asymptomatic period, and to score the effectiveness ofprophylactic therapeutics.

[0274] In order to determine whether TRF length is a useful marker indiagnosis of HIV infection, CD4⁺ cell counting was performed onasymptomatic HIV-infected individuals, and compared to TRF length,measured as discussed above. As shown above, peripheral lymphocytesstart with around 10 kb TRF length at birth, and reach a TRF length of5.0 at approximately age 120. The results were as follows:

[0275] A 30 year old HIV+ with a CD4 count of 476 had a TRF of 7.6.

[0276] A 46 year old HIV− control, had a TRF of 7.0.

[0277] A 34 year old HIV+ with a CD4 count of 336, had a TRF of 7.7.

[0278] A 46 year old HIV− control, had a TRF of 7.1.

[0279] A 32 year old HIV+ with a CD4 count of 448, had a TRF of 6.9.

[0280] A 33 year old HIV+ with a CD4 count of 358, had a TRF of 5.0(i.e., at a length observed for senescent cells)

[0281] The results indicate that the 33 year old HIV+ patient has asenescent telomere length in his CD4⁺ cells, which means that they areat the end of their replicative capacity. In contrast, the CD4⁺ countprovided no indication of the status of this patient. Indeed, onepatient actually had a lower CD4⁺ count.

[0282] Two weeks after the assay was performed, this patient experienceda precipitous drop in CD4⁺ count, going from 358 to 159, and wastherefore diagnosed AIDS, and rapidly acquired leukoplakia on thetongue. The other patients remain asymptomatic. Thus, this diagnosticprocedure is able to distinguish patients near the end of the course ofHIV infection, whereas the previously used marker (CD4⁺ count) couldnot.

[0283] The accelerated replicative senescence of CD4⁺ lymphocytes duringthe course of HIV infection provides an appropriate indication fortherapies designed to forestall telomere shortening, e.g., utilizing theCTO oligonucleotide described above. In addition, as described above,CD4⁺ cells of an individual at an early stage of infection can be bankedfor later administration to the individual. The efficacy of drugs, suchas AZT, may also be determined to study whether the drug slows the rateof proliferation of CD4⁺ cells, and is thus useful at all stages of thedisease. If not, it can be administered only when necessary during thecourse of the disease.

Example 9 Telomere Shortening in Human Mammary Epithelial (HME) Cells

[0284] Referring to FIG. 12, when digested with a restriction enzymehaving a 4-base recognition site (like Hinfl), most genomic DNA isdigested into small fragments. However, because the repetitive telomericsequences lack restriction sites, telomeres retain relatively largeterminal restriction fragments (TRFs) composed of 2-5 Kb of subtelomericDNA and age-dependent amounts of telomeric repeats. As previouslydescribed for human fibroblasts, lymphocytes and endothelial cells,telomere length shortens in normal human mammary epithelial cells duringin vitro cellular senescence (compare TRF length in lanes 1 (PDL 21) and2 (PDL 40)). In human mammary epithelial cells expressing E6 of humanpapilloma virus 16, the TRF length continues to shorten during theextended lifespan period until crisis and subsequently immortalizationoccurs (lane 3 (PDL 68)). The TRFs generally stabilize in immortalizedcells (lane 4 (PDL 81) and lane 5 (PDL 107)) consistent with there-expression of telomerase activity.

Example 10 Slowing Telomere Loss in Mammary Epithelial Cells Results inIncreased Replicative Lifespan

[0285] Normal human mammary epithelial cells can be established fromorganoids (obtained from reduction mammoplasty) and can be cultured indefined condition in a standard medium (MCDB170) devoid of serum.Epithelial cells with typical cobblestone morphology spread aroundorganoids plated in this medium. After the first subcultivation thesecultures enter a period of growth arrest for 2-3 weeks until apopulation of small, highly birefringent and rapidly dividing cellsexpand among larger cells. The medium (MCDB 104) apparently selects fora less differentiated cell type with increased growth potential. Thesecells can be subcultured for 40-45 additional doubling before undergoingcellular senescence.

[0286] As in Example 1, the change in proliferative lifespan and rate oftelomere shortening in cultured mammary epithelial cells treated withthe indicated amounts of CTO (occasionally referred to as C-RichTerminal Repeat (CTR)) versus control random oligonucleotides. Normalhuman mammary epithelial cells from a donor (31) were infected with theE6 gene of human papilloma virus 16. This gene product binds p53 proteinand permits HME31 cells to have extended life span by proliferating fromPDL 42 to PDL 62 when crisis occurs. During this extended lifespanperiod the TRFs shorten from an average of approximately 5 kb to 2.5 kb(compare in FIG. 12 HME31 PD 40 to HME31E6 PD 68).

[0287] As is demonstrated in FIG. 13, experiments initiated usingHME31E6 cells at PDL 36 were cultured in the presence of 3, 10, 30 and100 μM CTO. As controls the cells were cultured without oligonucleotides(nil) or with 30 μM random oligonucleotide. FIG. 13 demonstrates thatcompared to the nil control and the 30 μM random oligonucleotide, therewas a dose related retardation of TRF shortening between PDL 36 and 50.This is most easily seen by examining the subpopulation of telomere TRFsthat migrate more slowly than the rest, giving a discrete trailing band.Cells were maintained in logarithmic growth with medium changed andfresh oligonucleotide added three times per week.

[0288] Human mammary epithelial cells expressing HPV16 E6 bypass M1 andhave extended replicative lifespan. HME31 cells normally senesce at PDL42-45. When expressing E6 they will bypass Ml and divide until theyreach crisis (M2) at PDL 53-62. The TRFs in HME31 (E6) cells at PDL 40are approximately 5-6 Kb while at PDL 62 they are 3-4 Kb (see FIG. 12).As is demonstrated in FIG. 17, experiments initiated using HME31E6 cellsat PDL 36 were cultured in the presence of 30 μM and 100 μM CTR indefined medium without serum. As controls, the cells were culturedwithout oligonucleotide (control), or with a 30 μM randomoligonucleotide with the base content matched to the CTRoligonucleotide. FIG. 17 demonstrates that compared to the control andthe 30 μM random oligonucleotide, there was a dose-related extension ofthe replicative lifespan in cells treated with CTR oligonucleotides. Thecontrol cells divided approximately 20 times during the experiment,whereas the CTR-treated cells divided at least 40-50 times. Theseresults correlate well with the retardation of telomere shorteningobserved in FIG. 13.

Example 11 Extension of Life Span of IMR90 Fibroblasts

[0289] Referring to FIG. 14, IMR-90 lung fibroblasts TRFat PDL 30 weretreated with 10 μM, 30 μM or 100 μM phosphodiester CT0 or with onlymedia addition (control). The cells were cultured in medium containingregular defined supplemented calf serum. The cells were passaged in 24well dishes and subcultivated by trypsinization upon reaching confluencyat 25,000 cells per well. The cells were fed medium containingoligonucleotides at various concentrations daily. As a control, cellswere fed identical medium without oligonucleotides. As is illustrated inFIG. 14, there was approximately a 12-15% extension of total life spanwith CTO. In these experiments the control cells divided approximately15-18 times during the experiment, whereas the treated cells divided23-26 times. IMR-90 telomeres shorten approximately 50 b.p. per divisionand the TRF length of the control IMR-90 fibroblasts at senescence wasapproximately 9 kb. Since the 100 μM CTO-treated IMR-90 cells senescedat PDL 55, the predicted difference in the rate of TRF loss between thecontrol and the 100 μM CTO (9 kb vs 9.4 kb) is too small to be resolvedusing current techniques.

Example 12 GTO Experiments

[0290] As in Example 2, an immortalized human fibroblast cell line,IDH4, which has very short TRFs, was incubated with GTO oligonucleotide.Referring to FIGS. 15 and 16, cells were incubated in regular culturemedium containing serum in the presence of 10 μM, 30 μM and 100 μM GTO.The cells were fed fresh phosphodiester GTO oligonucleotide every otherday and subcultured when confluent for a total of 90 days. The cellswere still growing in GTO after 90 days at all concentrations used eventhough they grew more slowly at the higher GTO concentrations and wentthrough fewer population doubling (control, 45 PDL; 10 μM GTO 40 PDL; 30μM 35 PDL; 100 μM 25 PDL). When TRF analysis was performed after 90 daysthe IDH4 cells regained TRF length in a dose dependent manner with 30 AMand 100 AM being approximately the same (FIG. 15). This suggests thatthe presence of excess single-stranded TTAGGG DNA in the cell wasprobably influencing the feedback regulation of telomerase and actuallyincreasing telomerase activity and extending telomere length. Thecontrol and 30 μM GTO were passaged without oligonucleotide addition foran additional 90 days (approximately 35-40 PDL). As is illustrated inFIG. 16, the TRFs slowly shorten.

[0291] These data and those in Example 2, indicate that cell linesdiffer in their response to GTO oligonucleotide. Thus, prior to use ofsuch an oligonucleotide in therapeutic compositions it is important toensure that the target cells respond as desired. Should the effect seenabove occur, then the oligonucleotide should be chosen to change theresponse to that shown in Example 2. This can be done by choosing anoligonucleotide which binds to telomerase at a different site from thatbound by GTO. Applicant believes that the effect observed above iscaused by binding of GTO to required proteins, allowing telomerase to beactive to expand the telomeres. Thus, by choosing an oligonucleotidewhich does not bind such proteins the desired effect of reducingtelomerase activity can be achieved.

Example 13 Small Molecule Inhibition of Telomerase

[0292] The following is an example of a method for screening foractivity of small molecules as inhibitors of telomerase. Similarexamples will be evident to those in the art. Compounds that can bescreened include those which are not thought to be cytotoxic becausethey do not cause immediate cell death. Rather, such compounds act onlyafter several generations of inhibition of telomerase activity. Thus,previous drugs tested by standard means should now be retested todetermine their utility as claimed herein. Drugs which inhibittelomerase activity, or in some cases activate it in vivo (e.g.. at thelevel of transcription) are useful in treatment of disease are discussedherein.

[0293] We analyzed the effects of various nucleoside analogs, which arechain-terminating inhibitors of retroviral reverse transcriptases, onTetrahymena thermophila telomerase activity in vitro, and on telomerelength and maintenance, cell division and conjugation of Tetrahymenacells in vivo. In vitro assays of telomerase activity showed thatarabinofuranyl-guanosine triphosphate (Ara-GTP) and ddGTP were both veryefficient inhibitors of incorporation of labeled nucleotides intotelomeric DNA repeats, even at low inhibitor concentrations, whileazidothymidine triphosphate (AZT-TP), dideoxyinosine triphosphate(ddITP) or ddTTP were less efficient inhibitors of incorporation. All ofthese nucleoside triphosphate analogs, however, produced analog-specificalterations of the normal banding patterns seen upon gel electrophoresisof the synthesis products of telomerase, suggesting that the competitiveand/or chain terminating action differed at different positions alongthe RNA template.

[0294] The effects of these analogs in nucleoside form on Tetrahymenacell growth, conjugation, and telomere length were tested. Although celldivision rates and viability were unaffected after several weeks inculture with Ara-G, telomeres were consistently and rapidly shortened incultures containing AZT or Ara-G, and growth rates and viability of afraction of cells were decreased in AZT. In short-term experiments withcultures containing ddG, ddI, or 3′ deoxy-2′,3′-didehydrothymidine (d4T), d4T also showed shortened telomeres. ddG or ddI had no effect ontelomere length. AZT, Ara-G, Acycloguanosine (Acyclo-G), ddG and ddIwere added to conjugating cells, but none showed any irreversibledisruption of conjugation or macronuclear development, as shown byquantitation of the efficiency of formation of progeny cells. PCRanalysis of DNA from cells mated in AZT did show a decrease in theformation of 11 Kb rDNA, a marker for telomere addition duringMacronuclear developement.

[0295] The following materials and methods were used to obtain theseresults:

[0296]Tetrahymena thermophila strains SB210(VI) and PB9R(II), wherenumbers in parentheses indicate mating type, were maintained as stocksat room temperature in 1% PPYS (1% proteose peptone (Difco), 0.1% yeastextract (Difco) and 0.0015% Sequestrine (Ciba-Geigy)). Stocks werepassaged every three to four weeks.

[0297] For analysis of macronuclear DNA from cultures containing thenucleoside analog AZT (Sigma), or controls lacking analog, at varioustimepoints during vegetative divisions, cells from stationary stockcultures were inoculated into 25 ml thymine-deficient Iso-sensitestbroth (‘Isobroth’, Oxoid USA) in 250 ml flasks. Cultures were incubatedat 30° C. with shaking (100 rpm) for 48 hours. Cells were counted andplated at 1000 cells/1.5 ml in 24-well plates (Falcon) and grown at 30°C., without shaking, for 48 hours. 5 μl of these log phase cells wereused to inoculate 1 ml cultures (Isobroth) containing variedconcentrations of nucleoside analog. Thereafter, every 2-4 days cellswere transferred, either 5 μl per well, or 1-3 μl using a multi-prongedreplicator into fresh 1 ml broth containing AZT. Remaining cells werepelleted and stored at −80° C. until processed for DNA analysis.

[0298] For analysis of macronuclear DNA from vegetative culturescontaining the nucleoside analogs Ara-G (Calbiochem), ddG (Calbiochem),or ddI (Calbiochem), or controls lacking analog, stock cultures weregrown overnight in 2% PPYS as described. Cells were counted and platedat 100 cells/2 ml in 2% PPYS containing varied amounts of analog, 1%DMSO (Fisher) (as a control for ddG and Ara-G), or 2% PPYS alone. Cellswere replica plated into fresh medium every 2-6 days, and remainingcells were pelleted and stored at −80° C. until processed for DNAanalysis.

[0299] For analysis of macronuclear DNA from vegetative culturescontaining d4T (Sigma) or control lacking the analog, stock cultures(SB210 VI) were grown overnight in Isobroth as described. Cells werethen counted and duplicate cultures inoculated at 500 cells/5 mlIsobroth in 50 ml conical tubes, and grown at 30° C., shaking 80 rpm.500-2000 cells were transfered to fresh broth every 2-4 days, and theremainder pelleted and stored at −80° C. until processed for DNAanalysis.

[0300] For analysis of rDNA from cells conjugated in the presence ofnucleoside analogs, 50 ml overnight cultures (2% PPYS) were starved bypelleting cells and resuspending in an equal volume of Dryl's solutionbefore returning to 30° C. shaking (100 rpm) incubator for 18 hours.(1×Dryl's solution=0.5 g Na citrate, 0.16 g NaH₂PO₄H₂O, 0.14 g Na₂HPO₄per liter, plus 15 ml of 9.98 g CaCl₂.2H₂O/500 ml). Cells were thencounted and equal numbers mixed before pelleting (6 minutes in an IECtabletop centrifuge, ¾ speed), and resuspended in Dryl's to 1.5-2×10⁶/ml. Cells were plated at an average density of 1.5 cells/well into6-well plates (Falcon) and allowed to conjugate 6 hours, 30° C. withoutshaking. Mock-conjugated SB210 cells were treated identically but notmixed with PB9R cells. At 6 hours the cultures were checked for pairing(>90%, except SB210 controls) and either 1 ml Dryl's solution or 2% PPYScontaining the nucleoside analog (Acyclo-G purchased from Sigma) or noadded drug as control were added slowly with gentle swirling. Cultureswere returned to 30° C. for an additional 18 hours before beingharvested for DNA analysis.

[0301] For analysis of vegetative growth and macronuclear DNA fromsingle-cell cultures containing the nucleoside analogs AZT or Ara-G,SB210 (VI) cells were grown from stationary stock cultures overnight at30° C. with shaking (100 rpm) in 50 ml 2% PPYS or Isobroth. Cells werecounted and added to the appropriate medium plus analog (Ara-G to 1 mMor DMSO to 1% as control in 2% PPYS; AZT to 10 μM or 1 mM, or noaddition as control in Isobroth) and plated in 96-well plates (Falcon),100 μl per well at a density of 1 cell per well. 5 plates were preparedfor each analog or control. Wells were scored for cell growth and plateswere replica plated every 1-2 days (Ara-G and DMSO plates) or every 2-4days (AZT and Isobroth control plates) to maintain approximateinoculation densities of 1-10 cells per well for each passage.Occasionally individual wells were passaged by hand (1 μl inoculated perwell using a pipettor) into several blank wells, to expand the number oflive wells per plate as single-cell cultures were lost over time due tolow probability of being transferred at each passage. After passaging,cells were pooled, pelleted and stored at −80° C. until processed forDNA analysis.

[0302] Total cellular DNA was prepared essentially as described byLarson 50 Cell, 477, 1987, except that the Hoechst 33258-CsCl gradientpurification step was omitted.

[0303] Restriction digests, agarose gel electrophoresis, transfer of DNAto Nytran filters (Schleicher and Schuell), and hybridization with³²P-nick-translated or random-primed probes were carried out usingstandard procedures (Maniatis et.al. 1989). Telomere length was analyzedas described previously for Tetrahymena [Larson 50 Cell, 477, 1987].

[0304] For analysis of cycloheximide (CHX) sensitivity of cellsconjugated in the presence of analog, 50 ml cultures of each cell typewere grown overnight in 2% PPYS, starved in Dryl's for 18 hours, mated(5×10⁵cells/ml) for 6 hours, then analog was added. Cells were allowedto complete mating in the presence of the analog. Twenty-four hoursafter mixing, cells were diluted in Dryl's solution, counted and platedat 1 cell per well of 96-well plates in 1% PPYS without analog. Cellswere grown for 4 days in a humid chamber at 30° C., without shaking.Cells were then replica plated into 1% PPYS plus 15 μg/ml cycloheximide,allowed to grow for four days before scoring, and percent ofCHX-resistant wells was calculated. Because generation of progenyexpressing the cycloheximide marker requires successful production of anew macronucleus, cells whose macronuclear development was disrupted bythe analog are killed in CHX.

[0305] For PCR analysis of the llkb form of the rDNA from culturesconjugated in the presence of analog, 1.25 μM each of the telomericprimer (C₄A₂)₄ and a 25-mer rDNA primer (5′ GTGGCTTCACACAAAATCTAAGCGC3′) located 1371 nucleotides from the 5′ end of the rDNA were used in a“hot start” reaction containing 1 mM MgCl₂, 0.2 mM each dNTP, 1×PCRreaction buffer (Perkin Elmer Cetus), and 0.5 μl Amplitaq polymerase(Perkin Elmer Cetus). Sample DNA and polymerase were kept separate bythe use of Ampliwax PCR Gem 100 wax beads (Perkin Elmer Cetus),following manufacturer's instructions. The samples were heated to 95° C.for 1 minute, and then cycled 40 rounds in a Perkin-Elmer thermocycleras follows: 1 minute at 94° C., 30 seconds at 58° C., 3 minutes at 68°C. Identical reactions were done using 3′ micronuclear rDNA primers,9610 nucleotides from the 5′end, and (5′CAATAATGTATTAAAAATATGCTACTTATGCATTATC 3′), 10300 nucleotides from the 5′end.

[0306] Synthetic oligomers were prepared as described Greider 43 Cell,405, 1985. Extracts were prepared as described by Blackburn et.al., 31Genome 553, 1989.

[0307] A standard assay contained 50% by volume of heparin-agarosepurified telomerase, 25 μM TTP, 1.25 μM ³²P-labeled dGTP (400 Ci/mMol,Amersham), 1 μM oligo (either (T₂G₄)₄ or (T₂G₄)₂ mixed with water andheated at 90° C. for two minutes and cooled at 30° C. for 10 minutes),and 0.1 μl RNasin (40 U/ml, Promega) in a no-salt buffer.AZT-triphosphate was obtained from Burroughs Wellcome, N.C.Ara-G-triphosphate was purchased from Calbiochem and ddNTPs from Sigma.Reaction mixes were kept on ice until ready for use, and then mixed intotubes containing analog for incubation at 30° C. Reaction times werethirty minutes. Reaction rates under these conditions were determinedpreviously to be linear over time for thirty minutes. Identicalreactions were run without primers as controls. The reactions were thenprocessed essentially as described by Greider and Blackburn 337 Nature,331, 1989. For quantitative assays, aliquots of the reaction mixturewere spotted in triplicate onto DE81 paper and washed as describedGreider 43 Cell, 405, 1985. Incorporation of ³²P label from either³²P-TTP or ³²P-dGTP was measured to monitor the reaction rate. Forvisualization of the elongation reaction products, samples were heatedto 95° C. for 2 minutes and cooled on ice before loading onto a 12%polyacrylamide/8 M urea gel.

[0308] The model for the mechanism of the telomerase ribonucleoproteinenzyme from Tetrahymena is shown in FIG. 18A. The enzyme synthesizesTTGGGG repeats onto the 3′ end of a suitable DNA primer by copying atemplate sequence in the RNA moiety of the enzyme. For ease of referencein discussing the results, the residues in the template region arenumbered 1 to 9 (5′ to 3′ along the RNA). The standard telomerase assayused in this example consists of incorporation of dGTP and TTPsubstrates, one triphosphate ³²P-labeled, into synthesized DNA in thereaction shown in FIG. 18A. For the experiments discussed in thisexample we used as the DNA primer either 1 μM (T₂G₄)₄ or (T₂G₄)₂, underconditions in which the overall rate of incorporation of label wasdetermined previously to be linear over time. Incorporation of ³²P labelfrom either ³²P-TTP or ³²P-dGTP was measured to monitor the reactionrate, and the distributions of elongation products were analyzed bydenaturing polyacrylamide gel electrophoresis.

[0309] The effect of adding increasing amounts of AZT-triphosphate(AZT-TP) to the standard assay for telomerase activity is shown in FIG.19A. A series of control reactions using unlabeled TTP added at the sameconcentrations as the AZT-TP was run in parallel (FIG. 19A). Theunlabeled TTP inhibits incorporation of the ³²P-labeled TTP by simplecompetition. Quantitation of label incorporated into product in thisexperiment enabled us to determine the K_(m) for TTP to be −5 μM.Compared with addition of unlabeled TTP competitor, AZT-TP had only amodest quantitative effect on the incorporation of ³²P-labeled TTP (FIG.19A). Since AZT incorporation leads to chain termination, this resultindicates that AZT-triphosphate competes less efficiently for telomerasethan TTP. Similar results were obtained when incorporation of ³²P-dGTPwas monitored (FIG. 19B), with 50% inhibition occurring at −80 μMAZT-TP.

[0310] In similar experiments in which increasing concentrations ofarabinofuranyl-guanosine triphosphate (Ara-GTP) were added to thereaction, significant reduction of overall incorporation occurred evenat low concentrations of the analog (FIG. 19C). From parallelexperiments in which unlabeled dGTP was added as competitor (FIG. 19C),the K_(m) for dGTP under these reaction conditions was found to be 1-2μM. 50% inhibition occurred with 0.7 μM Ara-GTP; thus Ara-GTPpotentially competes as well as unlabeled dGTP for ³²P-dGTP. However, asincorporation of Ara-G causes chain termination, each Ara-G incorporatedis expected to have a greater impact on total incorporation thancompetition with unlabeled dGTP.

[0311] We also tested the effects of dideoxynucleoside triphosphates(ddNTPs) on the telomerase reaction. As shown previously for telomerase[Greider 43 Cell, 405, 1985], and as is the case for many other reversetrancriptases, ddNTPs are recognized by the enzyme and incorporated,causing chain termination with a subsequent shift in banding patternsand reduction of the average product length. Consistent with previousqualitative analyses of Tetrahymena and human telomerases [Greider 43Cell, 405, 1985; Morin 59 Cell, 521, 1989], ddGTP and ddTTP eachinhibited the incorporation of labeled ³²P-NTP into elongation products(FIG. 19D and E). ddGTP was a much more efficient inhibitor than ddTTP:under these reaction conditions 50% inhibition occurred at <0.1 and 5 μMddGTP and ddTTP respectively. As observed previously for Tetrahymenatelomerase [Greider 43 Cell, 405, 1985], no significant effects wereseen with either ddCTP or ddATP. In addition, ddITP inhibited telomerase(FIG. 19E), although less efficiently than ddGTP, with 50% inhibitionoccurring at 3 μM ddITP.

[0312] The size distribution of labeled products was then analyzed bydenaturing polyacrylamide gel electrophoresis. Consistent with theexpectation for a chain-terminator, the proportion of longer telomeraseproducts was decreased in the presence of AZT compared with cold TTPcompetitor controls (FIG. 20A; compare lanes 1 and 2 with lanes 3 to 5),and in the presence of Ara-G (FIG. 20A; lanes 7 and 8). Average productlength also decreased in the presence of Ara-GTP, ddGTP and ddITP (FIG.20A and B). In addition, each nucleoside triphosphate analog produceddistinctive and characteristic patterns of chain termination, as shownby analysis of the shifts in the banding patterns of the elongationproducts. With AZT-triphosphate, we saw increased relative intensitiesof the bands corresponding to the incorporation of T residues (copyingthe A residues at positions 2 and 3 on the template RNA (see FIG. 18A)).This change in banding pattern is consistent with simple chaintermination, which is predicted to increase the intensity of bandscorresponding to the position of both incorporated T residues. Similareffects were seen with ddTTP. We interpret this to mean thatAZT-triphosphate was recognized by the enzyme and incorporated into thecorrect positions in the growing telomeric sequence, causing chaintermination. However it cannot be excluded that the increase in relativeintensity of the band corresponding to position 3 on the template, whichprecedes addition of the second T, is also attributable to pausingcaused by competition with TTP and a slower reaction rate withAZT-triphosphate at position 2.

[0313] The results with Ara-GTP were also consistent with incorporationof Ara-G and consequent chain termination (FIG. 20A, lanes 7 and 8).Although there are four positions at which a G residue can beincorporated and therefore at which chain termination could occur, thestrongest increase was in the band corresponding to the G residuespecified by position 4, in the middle of the telomerase RNA template(see FIG. 18A). With ddGTP, chain termination appeared to occur mostefficiently at positions 6 and 5 (FIG. 20B, compare lane 1 with lanes 4to 6), and with ddITP, at position 5 (lanes 7 to 9).

[0314]FIG. 18B summarizes schematically the effects of the varioustriphosphate analogs on polymerization at each of the six positionsalong the template. There was no correlation between the efficiency of anucleoside analog as an inhibitor and the position of its maximal chaintermination on the template. For example, the potent inhibitors ddg-andAra-G-triphosphates cause maximal chain termination at differentpositions on the telomerase RNA template (5 and 6 for ddG, and 4 forAra-G).

[0315] In addition to nucleoside triphosphate analogs expected to act aschain terminators, we also tested rifampin, an inhibitor of bacterialRNA polymerase, and streptomycin sulfate. Streptomycin sulfate is knownto inhibit the activity of group I self-splicing introns at highconcentrations [von Ahsen 19 Nucl. Acids Res., 2261, 1991], and has aguanidino group that might be recognized by telomerase as part of theenzyme's specificity for G-rich DNA primers (Greider 51 Cell, 887,1987). Adding rifampin at concentrations up to 100 μg/ml did not affectthe quantitative incorporation of label or change the banding pattern ofthe elongation products. Streptomycin sulfate at 40 mM dramaticallyreduced the amount (FIG. 19F) and average length of elongation products,with little decrease in activity being seen in a 40 mM sodium sulfatecontrol. However, unlike the nucleoside triphosphate analogs, inhibitionby streptomycin did not appear to affect incorporation at specificpositions in the repeat. The inhibition by streptomycin may be usefulexperimentally as a criterion for telomerase activity in vitro. However,the significance of the inhibition by streptomycin is unclear, as it isdifficult to rule out that its effect is the result of nonspecificbinding to either the RNA moiety of telomerase or the DNA primer.

[0316] Because the triphosphate forms of the analogs AZT, Ara-G, ddT,ddG and ddI each inhibited (with varying efficiencies) telomerase invitro, we tested whether supplying each of these nucleoside analogs inthe cell growth medium caused in vivo changes in telomere length orsenescence. Additionally, Acyclo-G and d4T were tested on conjugatingand vegetative cells, respectively.

[0317] Previous work with Tetrahymena showed that at least onealteration of the telomerase RNA causes telomere shortening and cellularsenescence [Yu 344 Nature 126, 1990]. To test whether such a phenotypecould be produced by inhibitors of telomerase in Tetrahymena, duplicatelog-phase phase cultures were grown for prolonged periods in thepresence of varying concentrations of analogs. The growth and cellmorphology of these cultures were monitored, and DNA was isolated atdifferent times for telomere length analysis. AZT at 5 or 10 mM added toIsobroth medium strongly inhibited cell growth and killed cells within aday, and thus at these concentrations acted in a manner suggestive ofimmediate toxicity to cells, rather than of senescence. AZT added toIsobroth medium at lower concentrations (up to 1 mM) did not result insenescence of cultures maintained by subculture of −10³ cells pertransfer, over a 50-day period of continuous growth and subculturing ofthese cell cultures. From growth rate measurements it was calculatedthat the cells went through 150 to 250 cell generations in the course ofthis 50 day period. In similar mass transfer experiments no effects oncell doubling rate, morphology or long term viability were obtained withcells grown in 2% PPYS plus up to 2 mM Ara-G, the highest concentrationtested that did not cause immediate toxicity.

[0318] Telomere lengths in cells grown in the presence of the differentanalogs were monitored by Southern blot analysis of DNA samplesextracted at a series of time points during the subculturings. Thetelomeres of cells grown vegetatively in 1 and 5 mM AZT in 2% PPYSmedium were reproducibly shortened by up to an average of 170 base pairscompared with the control cultures grown in 2% PPYS in the absence ofthe drug (FIG. 21A and B). This shortening of telomeres occured in aconcentration-dependent manner (FIG. 21B), with at least 50% of themaximal shortening effect occurring by 10 μM AZT, the lowestconcentration tested. For each AZT concentration tested, the fulldecrease was seen within 3 days of culturing in the presence of the drug(15 to 30 cell divisions), but after this initial length adjustment, ateach drug concentration telomeres thereafter showed no statisticallysignificant shortening over time, and mean telomere length consistentlyremained static for at least 28 days of mass transfer subculturing.

[0319] Similar degrees and timing of telomere shortening were producedwith 1 or 2 mM Ara-G added to 2% PPYS culture medium (FIG. 21C). d4Tadded to Isobroth culture medium in concentrations ranging from 10 μM to1 mM produced shortened telomeres at 100 μM and 1 mM, again in aconcentration dependent manner, after 5 days (16 generations) inculture. In contrast, up to 1 mM ddG or ddI produced no changes intelomere length compared with control cultures, over a period of 5 daysof subculturing (15-20 cell generations) in 2% PPYS medium.

[0320] Because we had found previously that telomerase is stronglyinhibited in vitro by at least some of the analogs tested, and telomerelength is affected in vivo within an estimated 15 to 30 cell generationsby these analogs, it was possible that telomere addition was in factbeing disrupted in vivo, but that our failure to find any evidence ofprogressive telomere shortening or senescence was attributable to asubset of the cell population that escapes an inhibitory effect of theanalog on telomerase. We have shown previously that impairing telomerasein vivo by mutating the telomerase RNA produced senescence in mostcells, but only −10² single cell subclones were analyzed in theseexperiments, [Yu 344 Nature 126, 1990]. Under our mass transfersubculturing regime, in which about 10³ cells were transferred perpassage, if a fraction as small as −1% of the cells escaped senescence,and if their growth advantage was sufficiently high compared with cellslosing telomeres, they could become the predominant population in anycell passage and we would not have detected any phenotype.

[0321] To test whether we had missed such a subpopulation of cells, wecarried out the same experiments on vegetatively dividing Tetrahymenacells in the presence and absence of drug, but in these experiments thesubculturing was carried out by plating cells at an average of 1 to 10cells per well in microtiter plate wells in the presence of 10 μM and 1mM AZT, and 10 μM and 1 mM Ara-G. For each drug, cells were plated outin this manner for 30 consecutive days (90 to 150 cell generations) and16 consecutive days (50 to 80 cell generations) respectively for the 10μM and 1 mM drug concentrations. DNA was isolated at intervals fromcombined samplings of the wells for analysis of telomere length.

[0322] Compared with control medium lacking the nucleoside analog, nochanges in the plating efficiency were observed over the course of theexperiment for cells grown in 10 μM AZT and 10 μM or 1 mM Ara-G.However, in the presence of 1 mM AZT, monitoring growth rates of cellsmaintained in this way by single cell transfers allowed us to identifytwo general growth classes, which we designated as slow (0 to 1 celldoubling per day) and fast (2 to 4 cell doubling per day). The growthrate of fast cells was similar to that of the controls grown in Isobrothcontaining no AZT. Over time, the proportion of wells with slow cellsdecreased, as would be expected if they simply had a lower probabilityof being transferred, since they were present in lower cell densitiesthan fast cells, which grew to higher cell densities and for which thetiming of the plating protocols had been worked out. However, monitoringthe cells remaining in wells after transfers had been made from themshowed that the slow cells lost viability over time. In addition,throughout the course of the transfers, slow cells appeared fromformerly fast cell wells. We pooled cells from the slow growing wells(pooling of several microtiter wells was necessary to obtain sufficientDNA for Southern analysis) and compared their telomere lengthdistribution with that of pooled fast cells. The mean length and sizedistribution of telomeric DNA from pooled fast cells wereindistinguishable from those of control cells grown without AZT. Incontrast, the pooled slow cell DNA showed a slight decrease in meantelomere length and heterogeneity (FIG. 21D). Control cells grown inIsobroth medium had telomeres that were an average of 165 bp shorterthan cells grown in 1% PPYS medium. We believe that because thetelomeric G₄T₂repeat tracts in cells grown in Isobroth medium arealready markedly shorter than those of cells grown in the richer PPYSmedium, the additional amount of telomere shortening caused by growth in1 mM AZT is sufficient to reduce continually and stochastically afraction of the telomeres below a critical lower threshold required forfunction, thus causing the decreased viability of a subpopulation of thecells.

[0323] We examined the effects of AZT, Ara-G, Acyclo-G, ddI and ddG onprogeny formation by cells that have undergone conjugation. This processinvolves de novo formation of new macronuclear telomeres in the progenycells. Mracronuclear development in ciliated protozoans such asTetrahymena involves developmentally programmed, site-specificfragmentation of germline chromosomes into linear subchromosomes, whoseends are healed by de novo addition of telomeres. We showed previouslythat telomerase not only elongates pre-existing telomeres in vivo duringvegetative cell divisions [Yu 344 Nature, 126, 1990], but also functionsto directly add telomeric DNA onto non-telomeric sequences during thisdevelopmentally-controlled chromosome healing. Because of the immediaterequirement for telomere addition to fragmented DNA, it is possible thatthe latter process might be more sensitive to telomerase inhibition thantelomere maintenance during vegetative growth. To test whethernucleoside analogs cause inhibition of macronuclear development due to adisruption of telomere formation, we mated two strains of Tetrahymenawhich are sensitive to cycloheximide, but whose progeny after mating areresistant to cycloheximide. Synchronized mated cells were treated withAZT at concentrations ranging from 10 μM to 5 mM for a period beginningjust prior to when macronuclear development begins and continuing duringmacronuclear development (the period 6-24 hours after mating wasinitiated). At this point cells were diluted out in microtiter platewells in fresh medium lacking the analog, at an average cell density ofone cell per well, and allowed to grow for the minimum period beforeselection for cells that had successfully produced progeny. In attemptsto maximize the effect of AZT, cells were either refed at 6 hrs with 2%PPYS or Isobroth, or starved until 24 hrs (the duration of the AZTtreatment). Such starvation arrests macronuclear development at anintermediate stage. When refed, macronuclear development would then beforced to proceed in the presence of the AZT. Control, unmated parentalcells were also plated and exposed to drug. Similar experiments wereperformed with Ara-G, Acyclo-G, ddI and ddG. The results are shown inTable 4.

[0324] The control plates showed 99%-100% cell death in CHX, while themajority of cells that were mated with or without analog survived. Noneof the nucleoside analogs had any statistically significant effect onprogeny formation. The design of this experiment would prevent takeoverof the culture by a minority population that evaded the effects of thedrug, as described above. Therefore little or no irreversible disruptionof macronuclear development due to impaired telomerase activity andtelomere formation occurred in the presence of AZT, Ara-G, Acyclo-G,ddG, or ddI.

[0325] Although macronuclear development was not significantlydisrupted, analysis of the formation of a marker for telomere additionduring macronuclear development suggests that AZT reduces the efficiencyof telomere addition.

[0326] DNA from cells mated in the presence or absence of analog, andeither refed at 6 hours or starved fully for the duration of conjugationwere used in PCR with a telomeric primer and a 5′ rDNA primer. Thisselected for a fragment of the llkb rDNA to which telomeres had beenadded. The 11 kb rDNA is either a by-product of the 21 kb rDNA formedduring macronuclear development or an intermediate of this process. Itis present only transiently during new macronuclear development and assuch is a good marker for telomere addition in vivo. Knock-down ofrelative amounts of the 1400 nucleotide PCR-generated fragment from 11kb-rDNA was seen in DNA from cells conjugated in the presence of AZT,but not in those containing Ara-G, Acyclo-G, H₂O or DMSO controls or inmock-conjugated SB210 cells. To show that the DNA used in the PCRreactions was present and competent for PCR, identical reactions wererun using primers from the 3′-micronuclear copy of the rDNA. In allsamples the expected 810 nucleotide fragment was produced in substantialquantities (FIG. 22), indicating that the decrease in the 1400nucleotide telomere-containing PCR product in samples from cells matedin AZT is due to the presence of analog rather than contaminants in theDNA or reagents. Southern blotting with a 5′-rDNA probe confirmed thatthe telomere-containing PCR product was from the expected rDNA sequence,(FIG. 22B) and no cross-hybridization occurred to the 3′ PCR product. Anoverall decrease in telomere-containing PCR products was seen in allsamples that were re-fed at 6 hours post-mixing, but the decrease wasmore pronounced in samples that had been mated in the presence of AZT.TABLE 6 Effects of nucleoside analogs on progeny formation. CELLTREATMENT # CHX-R # TOTAL % CHX-R SB210 (NOT MATED) 1 215 0.5 PB9R (NOTMATED) 3 307 1 AZT (mM) 0 139 212 66 0.01 121 169 72 0.1 100 148 68 1.091 166 55 5.0 75 120 63 SB210 (NOT MATED) 0 57 0 AZT (mM) 0 165 214 770.01 67 92 73 0.1 128 190 67 1.0 60 125 48 5.0 89 168 53 1% DMSO 84 10977 ARA-G (mM) 0.01 114 141 81 0.1 134 167 80 1.0 89 161 55 1% DMSO 51 7568 ARA-G (mM) 1.0 51 86 59 2.0 40 92 43 SB210 (NOT MATED) 0 9 0 PB9R(NOT MATED) 0 37 0 ddI (mM) 0 63 75 84 0.001 59 71 83 0.01 85 96 89 0.183 106 78 1.0 100 110 91 1% DMSO 21 44 48 ddG (mM) 0.001 86 102 84 0.173 86 85 1.0 51 66 77 ACYCLO-G (mM) 0 36 45 80 0.017 80 107 75 0 78 11667 0.017 101 146 69

Example 14 G-Reaction for Reducing the Size of the Terminal RestrictionFragment

[0327] Human fibroblast DNA digested with restriction enzymes,electrophoresed, and hybridized by Southern blot makes possible theresolution of terminal restriction fragments (TRFs) which in turnreflect the relative length of telomeric repeat sequences (See FIG. 26,HinfI digested DNA, labeled “HinfI”; DNA not digested, labeled “O”).This Southern analysis is complicated by the fact that human and manyother species have long stretches of subtelomeric repetitive sequencesthat add to the TRF size. As a means of eliminating the artifactualinclusion of this subtelomeric repeats in a measurement of telomericrepeat length, a modified Maxam-Gilbert reaction is employed tohydrolyze the DNA at G residues. In the lane labeled “P only”(underloaded) the DNA is treated with piperidine in mild conditionswhich does not in itself decrease the size of the DNA. In the lanelabeled “P+DMS” the samples are pretreated with DMS. Not the substantialreduction in TRF size compared to the HinfI digest relecting thedeletion of subtelomeric sequences in the C-rich strand containing Gresidues. All lanes were probed with (TTAGGG)₃. This assay is thususeful for analysis of telomere lengths in diagnostic procedures.

Example 15 Fungal Telomeres

[0328] The following example illustrates various specific telomericsequences which can be used to identify specific fungi. Those in the artwill recognize that such sequences can be probed with oligonucleotidesto specifically diagnose the presence of a selected fungus. In addition,specific treatment of fungi can be effected by use of agents which bindto such sequences and reduce the long term viability of the fungal cell.

[0329] As described herein telomeric DNA is an attractive target forspecific drug therapy. Telomeres are short single-stranded protrusionswhich are accessible to specific drugs. Binding by such drugs willinterfere with normal telomere function and thus fungal cell viability.In similar experiments (routine to those in the art when conducted asdescribed herein) inhibitors or facilitators of such telomerereplication (or telomerase activity) can be discovered and used asanticancer, antiparasite and antifungal agents.

[0330] The significantly increased length of fungal telomeres makes themideal targets for antisense therapy or diagnosis. In addition, thisdifferent telomere structure indicates a different mechanism of actionof the telomerase, and thus its availability as a target for antifungalagents which are inactive on human or other animal cells.

[0331] Telomeric DNA sequences have generally been found to beremarkably conserved in evolution, typically consisting of repeated,very short sequence units containing clusters of G residues. Recentlyhowever the telomeric DNA of the budding yeast Candida albicans wasshown to consist of much longer repeat units. Here we report theidentification of seven additional new telomeric sequences from buddingyeasts. Although within the budding yeasts the telomeric sequences showmore phylogenetic diversity in length (8-25 bp), sequence andcomposition than has been seen previously throughout the wholephylogenetic range of other eukaryotes, we show that all the knownbudding yeast telomeric repeats contain a strikingly conserved 6 bpmotif of T and G residues resembling more typical telomeric sequences.We propose that G clusters in telomeres are conserved because ofconstraints imposed by their mode of synthesis, rather than by afundamental requirement for a specific common structural property oftelomeric DNA.

[0332] The DNA sequences of telomeres, the ends of eukaryoticchromosomes, have been found previously to be conserved even betweenvery diverse eukaryotes, typically consisting of tandem arrays of 5-8 bprepeating units characterized by clusters of G residues, producing amarked strand composition bias. However, the telomeric repeats of theopportunistic pathogen Candida albicans were shown to consist ofhomogeneous repeats of a 23 bp sequence that lacks any noticeable strandcomposition bias.

[0333] To determine the relationship of the apparently exceptional,complex telomeric repeat sequence of Candida albicans to the more usual,simple telomeric sequences, genomic DNA from budding yeast speciesrelated to both C. albicans and S. cerevisiae were analyzed by Southernblotting, using cloned C. albicans telomeric repeats as thehybridization probe. Under low-stringency hybridization conditions wedetected multiple cross-hybridizing bands in several species FIG. 28. Insome cases, the cross-hybridizing bands clearly were broad, acharacteristic feature of telomeric restriction fragments caused bydifferent numbers of telomeric repeats in individual telomeres among apopulation of cells.

[0334] Telomere-enriched libraries were constructed from genomic DNAfrom seven budding yeast species and strains. Telomeric clones wereidentified by their ability to hybridize to known yeast telomericrepeats (either the 23 bp C. albicans repeat or the TG₁₋₃ repeat of S.cerevisiae), or by screening for end-linked repetitive DNA sequenceswithout the use of a specific probe. Sequencing putative telomerefragment inserts from seven species identified clones that containedtandem repeats with unit lengths of 8-25 bp. With a single exception,the repeats showed no sequence variations within a species. In everycase the repeat array was present at the very end of the insert,directly abutting vector sequences, as would be expected for clonedtelomeres. The repeat-containing clone from each species hybridized backto the same pattern of restriction fragments observed originally withthe C. albicans or the S. cerevisiae probe used for library screening.Most of the bands were preferentially sensitive to Bal31 nuclease (FIG.29) indicating that the bulk of the repeat sequences are present at theends of chromosomes. The lengths of the tracts of repeats cloned fromthe different yeast species were typically between 250-600 bp, althoughthose from the two C. tropicalis strains were only 130-175 bp. That thisspecies has particularly short telomeres is also supported by their veryrapid loss during Bal31 digestion and by the relatively weakhybridization, even with species-specific telomere probes.

[0335]FIG. 30 shows an alignment of these newly discovered telomericrepeat unit sequences together with those of C. albicans and S.cerevisiae. Two striking features are apparent: the much greater varietyof the budding yeast telomeres, with respect to repeat unit lengths andsequence complexities, compared to other eukaryotes, and a conservedsix-base cluster of T and G residues that most resembles typicaltelomeric sequences.

[0336] The sequence relationships among the telomeric repeats aregenerally consistent with the phylogenetic relationships of thesebudding yeasts. The telomeric repeats of the two C. tropicalis strainsdiffer by only a single base polymorphism. The 25 bp telomeric repeatsof the closely related K. lactis and C. pseudotropicalis differ at onlyone position. The telomeric repeat sequences from C. albicans, C.maltosa, C. pseudotropicalis, C. tropicalis and K. lactis are 23-25 bpin length, with differences largely or entirely confined to the centralpart of the repeat. The 16 bp repeat unit from C. glabrata, the speciesin this study that may be most closely related to S. cerevisiae, is veryG-rich, which probably contributes to its cross-hybridization to theheterogeneous and smaller S. cerevisiae telomeric repeats. All thebudding yeast sequences, including the irregular S. cerevisiae repeats,have a perfect or ⅚ match to a 6 bp T/G sequence (boxed).

[0337] In the cloned telomere from C. tropicalis strain B-4414, we foundtwo telomeric repeat sequences that differed at the second base positionof the repeat, as shown in FIG. 30 repeat units in the B-4414 telomerewere homogeneous (and will be termed the “AC repeat”), but the remainingrepeat (henceforth termed the “AA repeat”) was identical to thehomogeneous telomeric repeats cloned from strain C. tropicalis B-4443.

[0338] To determine the distribution of these variant repeats among thetelomeres and strains of C. tropicalis, genomic DNA from several C.tropicalis strains including B-4414 and B-4443, and a control C.albicans strain were probed with oligonucleotide probes specific foreither the AA or the AC repeat (FIG. 31 left panel). Only strains B-4414and 1739-82, and to some extent the C. albicans telomeres, hybridizedwith the AC repeat-specific oligonucleotide probe (FIG. 31 left panel).However, genomic DNA from all of the C. tropicalis strains tested,including B-4443, but not from C. albicans, hybridized well with theoligonucleotide specific for “AA” repeats (FIG. 31 right panel). Theseresults clearly indicate that both B-4414 and 1739-82 contain at leasttwo forms of telomeric repeats, which are most likely variablyinterspersed in different telomeres, as signal rations with the twoprobes differed between individual telomeric fragments (FIG. 31A and B,lanes 1 and 2).

Example 16 Effects of Telomerase Inhibitors on Human Tumor Cell Growth

[0339] Agents that were shown to inhibit telomerase from Tetrahymenae.g., AZT, ddG, and ara-G were tested to determine their effect on humantelomerase activity, telomere repeat length, and cell growthimmortality. Of the compounds tested ddG and ara-G were effectiveinhibitors of human telomerase obtained from the tumor cell line 296.The data for ddG is shown in FIG. 27. The effect of the agents ontelomerase activity in intact cells was then studied utilizing thelymphoma cell line JY 616 which were maintained in RPMI 1640 with 0.25MHepes, 10% FCS, and penicillin/streptomycin (Gibco). The cells werecultured in 6-well plates (Falcon) with 5.0 ML of medium per well induplicate. Cells were passaged every 7-10 days which corresponded to 5-7mean population doublings (MPD), and seeded at 3×10⁴ cells per well intofresh medium containing analog or control. Cell viability was monitoredprior to harvest utilizing trypan blue stain (Gibco) during countingwith a hemocytometer. The average ratio of stained: unstained cells(dead:alive) was >90%. The intactness of the DNA was measured on aparallel gel by observing its mobility in a gel prior to digestion by arestriction enzyme.

[0340] As seen in FIG. 23, all JY cells grew in an immortal fashion inthe presence of a low concentration of the potential telomeraseinhibitors. At high concentrations (FIG. 24), the cells ceasedproliferating in the presence of 50 μM AZT and displayed a slowed growthin the presence of 20 μM ara-G. In support of the belief that thisinhibition of cell growth in the presence of 50 μM AZT, is due totelomerase inhibition, is the observation that the cells grew at anormal rate until week 3 and then ceased dividing. This is the effectone would expect if the inhibition of cell growth was via telomeraseinhibition (i.e., the cells require multiple rounds of cell division tolose their telomeric repeats). Also in support of the belief that AZTinhibited the growth of the cells via the inhibition of telomerase isthe finding shown in FIG. 25 where compared to week 1, and week 3 wherethe cells stopped dividing, the AZT treated cells had a marked decreasein mean telomere length compared to the control medium “R” at the sametime.

[0341] In addition, 10 μM ddG was shown to cause a decrease in telomerelength compared to the control (in this case a DMSO control). In FIG. 32it can be seen that JY cells studied in a manner similar to thatdescribed above, and treated with ddG, showed a markedly shortertelomere repeat length after 9 and 10 weeks compared to the DMSOcontrol. It should be noted that while JY cells are immortal, whencultured under the conditions described, they lose some telomericrepeats over 10 weeks. The addition of ddG markedly accelerated thisloss.

Example 17 Assay for Telomerase Inhibitors Utilizing Human Telomerase

[0342] The following is a micro assay for the rapid screening ofpotential inhibitors of human telomerase. The reaction consists of thefollowing components (given as a final concentration in 10 μl reactionvolume): 1×Human Telomerase Buffer (HTB; 50 mM Tris.Cl pH 7.5, 1 mMSpermidine, 5 mM β-mercaptoethanol (BME), 1 mM MgCl₂, 50 mM potassiumacetate (K-OAc), 2 mM DATP, dTTP, 0.625 μM [³²P]-dGTP (800 Ci/mmol, 10mCi/ml stock), 1 μM (TTAGGG)₃, 1 μl test compound, 5 μl of 1:5 diluted293 S-100 extract, and diethylpyrocarbonate(depc)-treated H₂O to 10 μl.

[0343] The HTB, dATP, dTTP, 293 S-100 diluted extract, oligo and depcH₂O are combined, and aliquots are distributed to individual wells of a96-well microtiter plate. Different concentrations of the test compoundare added to the wells and the plate is preincubated at 30° C. for 20minutes to allow for inhibition to occur. The [³²P]-dGTP is then addedto the wells, and the reactions are incubated at 30° C. for a further30-60 minutes. At the end of this time, duplicate aliquots are spottedonto DE81 filter paper and allowed to dry. The filter paper is thenwashed extensively in 0.5M Na₂HPO₄ to wash away the unincorporatedlabel, and briefly rinsed in water before drying. The filter is thenexposed to a phosphorimaging screen for 30-60 minutes, and the samplesare quantitated.

[0344] By comparing the sample signal to the RNase pretreated controlsand the no extract controls, a percent decrease in telomerase-generatedsignal can be calculated. Any sample causing a change in the amount ofradioactivity detected is electrophoresed on a DNA sequencing gel toconfirm inhibition and to observe the effect of the agent on telomeraseprocessivity. This will include determining if the inhibitory effect isa direct effect on the telomerase enzyme, or on one of the othercomponents in the reaction, by performing titrations of these componentsand observing the percent inhibition. When a direct in vitro effect ontelomerase is detected, the inhibitor is then be tested on culturedcells and in animal models such as tumor-bearing nude mice. As seen inFIG. 27, ddG was shown to inhibit human telomerase in a dose-dependentmanner. Similar results were obtained with ara-G.

[0345] Compositions

[0346] Compositions or products according to the invention mayconveniently be provided in the form of solutions suitable forparenteral or nasal or oral administration. In many cases, it will beconvenient to provide an agent in a single solution for administration.

[0347] If the agents are amphoteric they may be utilized as free bases,as acid addition salts or as metal salts. The salts must, of course, bepharmaceutically acceptable, and these will include metal salts,particularly alkali and alkaline earth metal salts, e.g., potassium orsodium salts. A wide variety of pharmaceutically acceptable acidaddition salts are available. These include those prepared from bothorganic and inorganic acids, preferably mineral acids. Typical acidswhich may be mentioned by way of example include citric, succinic,lactic, hydrochloric and hydrobromic acids. Such products are readilyprepared by procedures well known to those skilled in the art.

[0348] The agents (and inhibitors) of the invention will normally beprovided as parenteral compositions for injection or infusion. They can,for example, be suspended in an inert oil, suitably a vegetable oil suchas sesame, peanut, or olive oil. Alternatively, they can be suspended inan aqueous isotonic buffer solution at a pH of about 5.6 to 7.4. Usefulbuffers include sodium citrate-citric acid and sodiumphosphate-phosphoric acid.

[0349] The desired isotonicity may be accomplished using sodium chlorideor other pharmaceutically acceptable agents such as dextrose, boricacid, sodium tartrate, propylene glycol or other inorganic or organicsolutes. Sodium chloride is preferred particularly for bufferscontaining sodium ions.

[0350] If desired, solutions of the above compositions may be thickenedwith a thickening agent such as methyl cellulose. They may be preparedin emulsified form, either water in oil or oil in water. Any of a widevariety of pharmaceutically acceptable emulsifying agents may beemployed including, for example acacia powder, or an alkali polyetheralcohol sulfate or sulfonate such as a Triton.

[0351] The therapeutically useful compositions of the invention areprepared by mixing the ingredients following generally acceptedprocedures. For example, the selected components may be simply mixed ina blender or other standard device to produce a concentrated mixturewhich may then be adjusted to the final concentration and viscosity bythe addition of water or thickening agent and possibly a buffer tocontrol pH or an additional solute to control tonicity.

[0352] For use by the physician, the compositions will be provided indosage unit form containing an amount of agent which will be effectivein one or multiple doses to perform a desired function. As will berecognized by those in the field, an effective amount of therapeuticagent will vary with many factors including the age and weight of thepatient, the patient's physical condition, the blood sugar level to beobtained, and other factors.

[0353] Administration

[0354] Selected agents, e.g., oligonucleotide or ribozymes can beadministered prophylactically, or to patients suffering from a targetdisease, e.g., by exogenous delivery of the agent to an infected tissueby means of an appropriate delivery vehicle, e.g., a liposome, acontrolled release vehicle, by use of iontophoresis, electroporation orion paired molecules, or covalently attached adducts, and otherpharmacologically approved methods of delivery. Routes of administrationinclude intramuscular, aerosol, oral (tablet or pill form), topical,systemic, ocular, intraperitoneal and/or intrathecal. Expression vectorsfor immunization with ribozymes and/or delivery of oligonucleotides arealso suitable.

[0355] The specific delivery route of any selected agent will depend onthe use of the agent. Generally, a specific delivery program for eachagent will focus on naked agent uptake with regard to intracellularlocalization, followed by demonstration of efficacy. Alternatively,delivery to these same cells in an organ or tissue of an animal can bepursued. Uptake studies will include uptake assays to evaluate, e.g.,cellular oligonucleotide uptake, regardless of the delivery vehicle orstrategy. Such assays will also determine the intracellular localizationof the agent following uptake, ultimately establishing the requirementsfor maintenance of steady-state concentrations within the cellularcompartment containing the target sequence (nucleus and/or cytoplasm).Efficacy and cytotoxicity can then be tested. Toxicity will not onlyinclude cell viability but also cell function.

[0356] Some methods of delivery, e.g., for oligonucleotides, that may beused include:

[0357] a. encapsulation in liposomes,

[0358] b. transduction by retroviral vectors,

[0359] c. conjugation with cholesterol,

[0360] d. localization to nuclear compartment utilizing antigen bindingsite found on most snRNAs,

[0361] e. neutralization of charge of oligonucleotides by usingnucleotide derivatives, and

[0362] f. use of blood stem cells to distribute oligonucleotidesthroughout the body.

[0363] At least three types of delivery strategies are useful in thepresent invention, including: agent modifications, particle carrier drugdelivery vehicles, and retroviral expression vectors. Unmodified agentsmay be taken up by cells, albeit slowly. To enhance cellular uptake, theagent may be modified essentially at random, in ways which reduces itscharge but maintains specific functional groups. This results in amolecule which is able to diffuse across the cell membrane, thusremoving the permeability barrier.

[0364] Modification of agents to reduce charge is just one approach toenhance the cellular uptake of these larger molecules. The structuralrequirements necessary to maintain agent activity are well understood bythose in the art. These requirements are taken into consideration whendesigning modifications to enhance cellular delivery. The modificationsare also designed to reduce susceptibility to enzymatic degradation.Both of these characteristics should greatly improve the efficacy of theagent.

[0365] Chemical modifications of the phosphate backbone ofoligonucleotides will reduce the negative charge allowing free diffusionacross the membrane. This principle has been successfully demonstratedfor antisense DNA technology. In the body, maintenance of an externalconcentration will be necessary to drive the diffusion of the modifiedoligonucleotides into the cells of the tissue. Administration routeswhich allow the diseased tissue to be exposed to a transient highconcentration of the oligonucleotide, which is slowly dissipated bysystemic adsorption are preferred. Intravenous administration with adrug carrier designed to increase the circulation half-life of theoligonucleotides can be used. The size and composition of the drugcarrier restricts rapid clearance from the blood stream. The carrier,made to accumulate at the site of infection, can protect theoligonucleotides from degradative processes.

[0366] Drug delivery vehicles are effective for both systemic andtopical administration. They can be designed to serve as a slow releasereservoir, or to deliver their contents directly to the target cell. Anadvantage of using direct delivery drug vehicles is that multiplemolecules are delivered per uptake. Such vehicles have been shown toincrease the circulation half-life of drugs which would otherwise berapidly cleared from the blood stream. Some examples of such specializeddrug delivery vehicles which fall into this category are liposomes,hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesivemicrospheres.

[0367] From this category of delivery systems, liposomes are preferred.Liposomes increase intracellular stability, increase uptake efficiencyand improve biological activity.

[0368] Liposomes are hollow spherical vesicles composed of lipidsarranged in a similar fashion as those lipids which make up the cellmembrane. They have an internal aqueous space for entrapping watersoluble compounds and range in size from 0.05 to several microns indiameter. Several studies have shown that liposomes can deliver agentsto cells and that the agent remains biologically active.

[0369] For example, a liposome delivery vehicle originally designed as aresearch tool, Lipofectin, has been shown to deliver intact mRNAmolecules to cells yielding production of the corresponding protein.

[0370] Liposomes offer several advantages: They are non-toxic andbiodegradable in composition; they display long circulation half-lives;and recognition molecules can be readily attached to their surface fortargeting to tissues. Finally, cost effective manufacture ofliposome-based pharmaceuticals, either in a liquid suspension orlyophilized product, has demonstrated the viability of this technologyas an acceptable drug delivery system.

[0371] Other controlled release drug delivery systems, such asnanoparticles and hydrogels may be potential delivery vehicles for anagent. These carriers have been developed for chemotherapeutic agentsand protein-based pharmaceuticals.

[0372] Topical administration of agents is advantageous since it allowslocalized concentration at the site of administration with minimalsystemic adsorption. This simplifies the delivery strategy of the agentto the disease site and reduces the extent of toxicologicalcharacterization. Furthermore, the amount of material to be applied isfar less than that required for other administration routes. Effectivedelivery requires the agent to diffuse into the infected cells. Chemicalmodification of the agent to neutralize negative or positive charges maybe all that is required for penetration. However, in the event thatcharge neutralization is insufficient, the modified agent can beco-formulated with permeability enhancers, such as Azone or oleic acid,in a liposome. The liposomes can either represent a slow releasepresentation vehicle in which the modified agent and permeabilityenhancer transfer from the liposome into the targeted cell, or theliposome phospholipids can participate directly with the modified agentand permeability enhancer in facilitating cellular delivery. In somecases, both the agent and permeability enhancer can be formulated into asuppository formulation for slow release.

[0373] Agents may also be systemically administered. Systemic absorptionrefers to the accumulation of drugs in the blood stream followed bydistribution throughout the entire body. Administration routes whichlead to systemic absorption include: intravenous, subcutaneous,intraperitoneal, intranasal, intrathecal and ophthalmic. Each of theseadministration routes expose the agent to an accessible diseased orother tissue. Subcutaneous administration drains into a localized lymphnode which proceeds through the lymphatic network into the circulation.The rate of entry into the circulation has been shown to be a functionof molecular weight or size. The use of a liposome or other drug carrierlocalizes the agent at the lymph node. The agent can be modified todiffuse into the cell, or the liposome can directly participate in thedelivery of either the unmodified or modified agent to the cell.

[0374] Most preferred delivery methods include liposomes (10-400 nm),hydrogels, controlled-release polymers, microinjection orelectroporation (for ex vivo treatments) and other pharmaceuticallyapplicable vehicles. The dosage will depend upon the disease indicationand the route of administration but should be between 10-2000 mg/kg ofbody weight/day. The duration of treatment will extend through thecourse of the disease symptoms, usually at least 14-16 days and possiblycontinuously. Multiple daily doses are anticipated for topicalapplications, ocular applications and vaginal applications. The numberof doses will depend upon disease delivery vehicle and efficacy datafrom clinical trials.

[0375] Establishment of therapeutic levels of agent within the targetcell is dependent upon the rate of uptake and degradation. Decreasingthe degree of degradation will prolong the intracellular half-life ofthe agent. Thus, chemically modified agents, e.g., oligonucleotides withmodification of the phosphate backbone, or capping of the 5′ and 3′ endsof the oligonucleotides with nucleotide analogues may require differentdosaging.

[0376] It is evident from the above results, that by modulatingtelomerase activity and monitoring telomere length and telomeraseactivity, one may provide therapies for proliferative diseases andmonitor the presence of neoplastic cells and/or proliferative capacityof cells, where one is interested in regeneration of particular celltypes. Assays are provided which allow for the determination of bothtelomere length, particularly as an average of a cellular population, ortelomerase activity of a cellular population. This information may thenbe used in diagnosing diseases, predicting outcomes, and providing forparticular therapies.

[0377] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

[0378] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

Example 18 An Alternative Method of Measuring Telomere Repeat Length

[0379] An alternative method to measure telomere length exploits thefact that the telomere sequence lacks guanine residues in the C-richstrand. Unmelted genomic DNA can be mixed with a biotinylatedoligonucleotide containing the sequence Biotinyl-X-CCCTAACCCTAA whichwill anneal to the single stranded G-rich overhang, followed byextension with the Klenow fragment of DNA polymerase in the presence ofdTTP, DATP and radioactive dCTP. The DNA is then mixed withstreptavidin-coated magnetic beads, and the DNA-biotin-streptavidincomplexes recovered with a magnet. This procedure purifies the telomeresand the radioactivity recovered at this step is proportional to thenumber of telomeres. The DNA is then melted, and DNA synthesis primedwith fresh CCCTAACCCTAA oligonucleotide, dTTP, dATP and radioactivedCTP. The radioactivity incorporated during this step is proportional tothe number of telomeric repeats (telomere length) after correction forthe number of telomeres present as determined during the first step.This value can then be converted into an actual telomere length bycomparison to a standard curve generated from telomeres of previouslydetermined lengths.

Example 19 An Alternative Method to Isolate Telomeric Sequences

[0380] Large telomeric DNA is purified as follows. A biotinylatedoligonucleotide with the sequence biotinyl-X-CCCTAACCCTAA is used toprime DNA synthesis in double-stranded DNA. The only sequences withwhich this oligonucleotide can anneal will be the single-stranded baseoverhangs as telomere ends. The extended DNA, which now has a morestable structure than that provided by the initial 12 bp overlap, isthen recovered using streptavidin. For large DNA, the DNA could bedigested with a rare-cutting restriction endonuclease such as Not1, thensubjected to pulse-field electrophoresis, Streptavidin, covalentlyattached to a block of agarose near the origin, would bind to thebiotinylated DNA and restrict the migration of the telomeres whilepermitting the bulk of genomic DNA to migrate into the gel. TelomericDNA could then be recovered, cloned and characterized.

[0381] Alternately, smaller telomeric DNA fragments are recovered fromsheared DNA using streptavidin coated magnetic beads. The followingmethod was used to obtain these results:

[0382] Pilot experiments had indicated that the shearing forcesgenerated during the mixing and separation procedure yielded DNAfragments approximately 20 kbp long. In order to maximize the amount ofsubtelomeric DNA obtained, DNA from a T-antigen immortalized cell line(IDH4, derived from IMR90 human lung fibroblasts) that had very fewtelomeric repeats (short TRFs) were used as the source of the DNA. 50 μgof IDH4 DNA was mixed with 1.25 pmol of biotinylated CCCTAACCCTAAprimer, 33 μM each of dATP, dTTP and dCTP, and 2 U of the Klenowfragment of DNA polymerase, in a final volume of 100 μl of BoehringerMannheim restriction endonuclease Buffer A and extended for three hoursat 37° C. A similar amount of a biotinylated TTAGGGTTAGGG primer (whichshould not anneal to the G-rich telomeric overhang) was added to asecond reaction as a negative control. Five μl of M-280Streptavidin-coated magnetic beads (Dynal, Inc.) were then added andgently mixed for 2 hours at room temperature, then biotinylatedDNA-streptavidin-bead complexes were recovered by holding a magnetagainst the side of the tube, and washed first with isotonic salinecontaining 0.1% Triton X-100 and 0.1% bovine serum albumin, and thenwith Sau3a restriction enzyme digestion buffer. The DNA was thensuspended in 20 μl Sau3a digestion buffer (New England Biolabs) anddigested with 3 U of Sau3a in order to release the subtelomeric DNA,leaving the terminal restriction fragments attached to the beads. Thebead-TRF complexes were removed with a magnet, and the supernatantcontaining the subtelomeric DNA was heated at 70° C. for one hour toinactivate the Sau3a. PCR linkers were added to the subtelomeric DNAfragments by adjusting the buffer to 5 mM DTT and 0.5 mM ATP, adding 25pmol annealed PCR linkers plus 1.5 U of T4 DNA ligase, and incubatingovernight at 16° C. The sequence of the PCR linkers used is:

[0383] OLM2: 5′ TGGTACCGTCGAAAGCTTGACTG 3′

[0384] DMO1: 3′ ATGAACTGACCTAG 5′

[0385] These linkers are designed such that the annealed linkers have aSau3a compatible end (5′ GATC 3′), the 3′ end of OLM2 will becomeligated to the subtelomeric DNA fragment, while the 5′ end of DMO1(which is not phosphorylated) will remain unligated. The overlap betweenOLM2 and DMO1 has an approximate melting point of 24° C., so thatheating the ligated mixture to 70° C. for 20 minutes both inactivatesthe ligase and dissociates DMO1. Half of the ligation mix was thendiluted in PCR buffer with 100 pmol OLM2/100 μl as the only primer.After three thermal cycles of 72° C.×1 min then 85° C.×1 min (in orderto fill in the complementary sequence to OLM2 before melting the DNA)the DNA was PCR amplified for 20 cycles (94° C.×1 min, 55° C.×1 min, 72°C.×3 min).

[0386] The purity of the PCR amplified subtelomeric library was assessedby in situ hybridization to metaphase chromosomes. Three probes wereprepared by amplifying the libraries in the presence of digoxigeninlabelled UTP: a positive control in which PCR linkers had been ligatedto a concatenated TTAGGG oligonucleotide to produce an amplified mixturecontaining an average size of about 1 kbp of telomeric repeats(“Concatenated GTR”); a negative control of the DNA selected with thebiotinylated TTAGGGTTAGGG primer (“GTR-selected”); and the experimentallibrary selected with the biotinylated CCCTAACCCTAA primer(“CTR-selected”). The slides were hybridized to the different probes,stained with an anti-digoxigenin monoclonal antibody followed by analkaline phosphatase conjugated anti-mouse antibody, then coded andscored for the presence of signal at internal sites versus telomericends. Only after being analyzed was the code broken. The results areshown in Table 7: TABLE 7 In Situ Hybridization Analysis of SubtelomericDNA (two experiments) Internal Probe End Signal Signal % TelomericConcatenated 104, 46 20, 19 81%, 71% GTR GTR-selected 20, 32 90, 95 18%,25% CTR-selected 76, 79 57, 29 57%, 73%

[0387] The CTR-selected PCR amplification products were then cloned, and37 individual clones were picked and analyzed by in situ hybridization.10/37 (27%) of these clones gave telomeric signals. The reason why amuch smaller fraction of the individual clones were telomeric than thefraction of signals in Table 7 is due to the complexity of the PCRamplified material: Actual telomeric DNA would be relatively abundantand thus be able to give a signal, while contaminating internalsequences would be highly diverse and thus each individual sequence inthe mixture would tend to be too rare to give a signal. The 20 kbp ofDNA at the end of each of 46 chromosome ends represents approximately1/3000 of the genome. The telomeric location of approximately ⅓ of thecloned CTR-enriched DNA thus indicates that using the biotinylated CTRresulted in a 1000-fold enrichment for telomeric DNA.

[0388] Seven of the telomeric clones were present on individualtelomeres, while three hybridized to multiple telomeres. Thecharacteristics of the ten telomeric clones are listed in Table 8, andpartial DNA sequences from all but clone CSITU6 are shown in Table 9.TABLE 8 Characteristics of Subtelomeric Clones Number of Clone Approx.Size Telomeric Signals CSITU5 1.5 Kbp single CSITU6 0.5 Kbp multipleCSITU9 0.9 Kbp single CSITU13 0.9 Kbp single CSITU22 0.9 Kbp multipleCSITU24 0.9 Kbp multiple CSITU33 0.8 Kbp single CSITU37 0.9 Kbp singleCSITU38 0.9 Kbp single CSITU51 1.5 Kbp single

[0389] TABLE 9 Sequences of Subtelomeric 5a Clones CSITU5   1GATCTAGGCACAGCTGCTTCTCATTAGGCAGGTCTCAGCTAGAAGACCAC  51TTCCCTCCCTGAGGAAGTCAACCCTTCTGCCACCCCATGGCCTTGCTTAA 101TTTTCAGACTGTCGAATTGGAATCCTACCTCCATTAGCTACTAGCTTGGG 151CAAGATACAGAGCCCTCCC Total number of bases is: 169 DNA sequencecomposition: 39 A; 54 C; 33 G; 43T; 0 OTHER CSITU9   1ATATATGCGCTACATAAATGTATCTAGATGCAATTATCTAGATACATATA  51AGAAAGTATTTGAAGGCCTTCTACAAGGCTTAGTTATTATATTGGTTCAT 101 ACAAGTTCTTCTTCAGTotal number of bases is: 116 DNA sequence composition: 39 A; 17 C, 18G; 42 T; 0 Other CSITU13   1ATCCTTCTCCGCAAACTAACAGGAACAGAAAACCAAACACTGCATGTTCT  51CACATCATTGTGGGAGTTGAACAATGAGAACACATGGACACAGGGAGGGG 101AACATCACACACTCGGGGTGTCAGCCGGGTGGGAGGGTAGAGGAGGAGAA 151ATACCTAAGTTCCAGATGACAGGTTG Total number of bases is: 176 DNA sequencecomposition: 58 A; 37 C; 50 G; 31 T; 0 Other CSITU22   1GATCTATGCTACCTCTAGGGATGGCACCATTCACAAGCACAAAGGAGATG  51TCAGTGATTAAAAACACATGCTCTGGAGTCTGAGAGACTTTGACACTTGC 101TAGCTTGTGGACTCTAGAGTTTAAGGTATCTGGACCCCTTTTTTCCCTCA 151TGTGCATAATGAAGAGATT Total number of bases is: 169 DNA sequencecomposition: 47 A; 35 C; 39 G; 48 T; 0 Other CSITU24   1GATCAACACTGTTAGTTGAGTACCCACATCACAAACGTGATTCTCAAAT  51GCCTTCCTTCCTGTCTAGTTTCTATAGGTATATATTTCCTTTTTCGCAT 101AGGCCTGAAAAGCCGCCTCCAAATGCCGCCTTCCAGACACTATAAAAAG 151AGGGTTCAAACCTACTCTATGAAAGGGAATGTTCAACACAGA Total number of bases is: 192DNA sequence composition: 58 A; 49 C; 33G; 52 T; 0 Other CSITU33   1GATCTGTTTATTATTCTTCCAATATCTCCCCATCTCTTAAAAATTGGTTA  51TTTCTTCGTTCATACATTTTTATCTCCCAAATTANNNNTGAGACTGGTTT 101GAAGAGAGGAAAGCAATGTACACACTTTTATATTCCACCATGTATATCCG 151 GATATCC Totalnumber of bases is: 157 DNA sequence composition: 43 A; 32 C; 19 G; 59T; 4 Other CSITU37   1 AATCCTCCTACCCCTCCCTTTGTTAGCCTGCCATTACAGGTGTGAG 51 CCACCATTGCTCATTCGTCCGTTTTCATTCAACAATCATCCATCTA 101TTACATGTGAGGGATCTTCAGGTCATGGAAATTC Total number of bases is: 135 DNAsequence composition: 32 A; 37 C; 22 G; 44 T; 0 Other CSITU38   1GATCACTTGAGCCCAGGAGTTTGAGACCAGCCTGGGTGACATGGCAAAAC  51CCCATCTCTACCAAAAGAAAAAAANNNNACAAATTGGTGGTGTTGATGGT 101 CGGCGACCATTGATCCCTotal number of bases is: 117 DNA sequence composition: 35 A; 27 C; 28G; 23 T; 4 Other CSITU51   1GATCAGGGAGGGGCCGAAAACTGGAGATGCAGGTGTGCTGTAAGACACTG  51CAGAGAGGGCATTTACCTGCCCCATCATCCAGCACAGGAACAGCGACTGA 101CAGCGCTCACCCACCCACCATCGCCAGTCACACTGGG Total number of bases is: 137 DNAsequence composition: 37 A; 42 C; 39 G; 19 T; 0 Other

[0390] The CTR-enriched subtelomeric PCR amplified library has also beenused to screen a cDNA library. 32 clones have been isolated, and partialsequence has been obtained form five clones. Their sequences are shownin Table 10.

[0391] Two of these clones, PhC4 and PHCS, have been characterized onNorthern blots. Both hybridize to the same two mRNAs of approximately6.2 and 7.7 Kb. Since the 3′ sequences of PHC4 and PHC5 are different,this suggests they may represent alternative splicing products of thesame gene. Both messages are abundant in PDL 38 IMR90 cells, which haverelatively long telomeres, and neither is expressed in the immortal IDH4cells (which have very short telomeres) that were derived from IMR90.This supports the hypothesis that the expression of genes located in thesubtelomeric DNA are regulated by telomeric length. This data isevidence that the above mentioned procedure provides a means ofobtaining sequences located in the proximity of telomeres, some of whichencode mRNA. Those sequences which are unique to individual chromosomeswill be useful in genomic mapping. Those which are active genes anddifferentially expressed in cells with differing telomere length, mayplay an important role in communicating information relating to telomerelength to the cell. Genes that regulate the onset of M1 senescence canbe isolated by these means, as will as genes which modulate telomeraseactivity. The function of the telomeric genes can be identified byoverexpression and knock-out in young senescent and immortal cells. SuchcDNAs, antisense molecules, and the encoded proteins may have importanttherapeutic and diagnostic value in regard to their modulation of cellproliferation in age-related disease and hyperplasias such as cancer.TABLE 10 Partial Sequence of subtelorneric cDNA clones. PHC4-5′ end  1GGCTCGAGAACGGGAGGAGGGGGCTCTTGTATCAGGGCCCGTTGTCACAT 51CTGCTCTCAGCTTGTTGAAAACTCATAATC Total number of bases is: 80 DNA sequencecomposition: 17 A; 19 0; 24 G; 20 T; 0 Other PHC5-3′END   1AGGTCCCTTGGTCGTGATCCGGGAAGGGGCCTGACGTTGCGGGAGATCGA  51GTTTTCTGTGGGCTTGGGGAACCTCTCACGTTGCTGTGTCCTGGTGAGCA 101GCCCGGACCAATAAACCTGCTTTTCTTAAAGGAAAAAAAAAAAAAAAAAA 151 AAAAAAA Totalnumber of bases is: 157 DNA sequence composition: 47 A; 31 C; 44 G; 35T; 0 Other PHC 7   1 ATCTAGGTTTTTTAAAAAAGCTTTGAGAGGTAATTACTTGCATATGAGAG 51 AATAAAACATTTGGCACATTGTTAAAAAAAAAAAAAAAAAAAAAAAAAAA 101AAAAAAAAAAAAAAAAAAAA Total number of bases is: 120 DNA sequencecomposition: 73 A; 7 C; 14 G; 26 T; 0 Other PHC8   1CTCATTTACTTTTCTCTTATAGCGTGGCTTTAAACATATATACATTTGTA  51TATATGTATATATGAATATAATGTATAAAATGTATGTAGATGTATATACA 101AAAAATAAACGAGATGGGTTAAAGATATGTAAAAAAAAAAAAAAAAAAAA Total number of basesis: 149 DNA sequence composition: 69 A; 11 C; 19 G; 50 T; 0 Other PHC9  1 AGTCCCAGCTACTCGGGAGGGCTGAGGCAGGAGAATGGCGTGAACCCAGG  51AGGCGAAGCTTGCAGTGAGCTGAGATCGCGCCACTGCACTCCAGCCTGGA 101CGACAGAGCGAGACTCTGTCTCAAAAAAAAAAAAAAAAAAAA Total number of bases is: 169DNA sequence composition: 47 A; 35 C; 39 G; 48T; 0 Other

Example 20 Isolation of Factors that Derepress Telomerase

[0392] The M2 mechanism of cellular senescence occurs when insufficientnumbers of telomeric repeats remain to support continued cellularproliferation. Escape from the M2 mechanism and immortalization occurconcomitantly with the induction of telomerase activity andstabilization of telomere length, and thus the inactivation of the M2mechanism directly or indirectly derepresses telomerase.

[0393] The gene(s) regulating the M2 mechanism have been tagged withretroviral sequences. The methods by which this was accomplishedconsisted of first determining the frequency at which a clone of SV40T-antigen transfected human lung fibroblasts was able to escape M2 andbecome immortal (T-antigen blocks the Ml mechanism, thus the M2mechanism is the sole remaining block to immortality in these cells).The pre-crisis cells were then infected with a defective retrovirus inorder to insertionally mutagenize potential M2 genes, and it was shownthat the frequency of immortalization was increased by almostthree-fold. Finally, pulse-field electrophoresis of differentimmortalized insertionally mutagenized lines was used to identify whichof the lines became immortal due to an insertion into the same M2 gene.Since an M2 mechanism gene has now been tagged with retroviralsequences, those with ordinary skills in the art can now clone andidentify the specific gene. The methods used were as follows:

[0394] The frequency of escape from crisis (e.g., the immortalizationfrequency of T-antigen expressing cells) was estimated using an approachbased on what is essentially a fluctuation analysis as previouslydescribed (Shay, J. W., and Wright, W. E. (1989) Exp. Cell Res. 184,109-118). SW26 cells (a clone isolated from IMR 90 normal human lungfibroblasts transfected with a vector expressing SV40 large T antigen)were expanded approximately 15 PDL's before crisis into multiple seriesat a constant cell density of 6667 cells/cm². Each series wassubsequently maintained as a separate culture, so that at the end of theexperiment the fraction of each series that gave rise to immortal celllines could be determined. Cultures were split at or just prior toconfluence at 6667 cells/cm². Once cells reached crisis they were splitat least once every three weeks until virtually no surviving cellsremained or the culture had immortalized. When too few cells wereobtained, all of the cells were put back into culture in a single dish.Fibroblasts were considered immortal if vigorous growth occurred aftercrisis during two subcultivations in which 1000 cells were seeded into50 cm² dishes and allowed to proliferate for three weeks for each cycle.

[0395] SW 26 cells enter crisis at approximately PDL 82-85. Numerousvials of SW26 cells (8×10⁶ cells/vial) were frozen at PDL 71, andtesting verified that spontaneous immortalization events had not yetoccurred. Five vials were thawed, scaled up for 4 days to approximately10⁸ cells (thus to approximately PDL 74), than trypsinized and combinedinto a single pool of cells in 40 ml of medium and distributed into 20010 cm² dishes. Thirty dishes were treated with 25 μg/ml bleomycinsulfate (a chemical mutagen) for two hours in serum free medium one daylater. Since this concentration of bleomycin sulfate resulted inapproximately 50% of the IMR-90 SW26 cells dying, these dishes had beenplated at twice the cell density as the rest.

[0396] The remainder of the dishes were used as controls (70 dishes) orinfected with LNL6 defective retrovirus (100 dishes). LML6 was generatedin the amphotrophic packaging line PA317 according to previouslydescribed procedures (Miller and Rosman, 1989, Biotechniques 7,980-990). Culture supernatant from LNL6 infected PA317 cells were usedto infect one hundred dishes containing approximately 5×10⁵ cells in thepresence of 2 μg/ml of polybrene. Control medium supernatant fromuninfected PA317 cells containing polybrene were used to treat 70 dishesand served as controls. Within a few days after infection, all controland experimental dishes were counted and each dish contained 1-2×10⁶cells. The PDL of each dish was calculated and cells were then replatedat 0.33×10⁶ in 50 cm² dishes and maintained separately to conduct thefluctuation analysis.

[0397] Bleomycin treated SW26 cells escaped crisis with an approximatelytwo-fold higher frequency (7.7×10⁻⁷) than the spontaneous rate(4.7×10⁻⁷). Pre-crisis SW26 cells infected with the defective retrovirusLNL6 in order to produce insertional mutations yielded a frequency ofescape from crisis (10.9×10⁻⁷) that was 2-3 fold greater than the ratefrom simultaneous control series mock-infected with culture supernatantfrom the non-infected packaging line. TABLE 10 Bleomycin SulfateExrosure and Retrovirus Infection Increase Immortalization FrequencyAddition Immortalization Frequency Nil 10/68 4.4 × 10⁻⁷ Bleomycin  7/277.7 × 10⁻⁷ sulfate LNL6 retrovirus 36/99 10.9 × 10⁻⁷ 

[0398] DNA has been isolated from 23 of the 36 independent cell linesobtained following insertional mutagenesis with LNL6, and 7 of these(30%) did not contain retroviral sequences when analyzed on SouthernBlots, while most of the remainder contained single insertions. Giventhat those without retroviral insertions had to represent spontaneousimmortalization events, most of the remaining clones with retroviralinsertions should be due to insertional mutagenesis if the frequency ofimmortalization was actually increased 2-3 fold. DNA from 12 lines hasbeen digested with the rare-cutting enzyme Sfi1, followed by pulse-fieldelectrophoresis, transfer to nylon membranes and probing with theretrovirus LTR. Six of the 12 lines contained a common band ofapproximately 350 Kbp that hybridized to the retroviral LTR. Four ofthese six have also been analyzed following BamHl digestion, and threeof these four also contained a common band of approximately 20 Kbp.Given that the retrovirus is 6 Kbp long, this strongly suggests that theretrovirus has inserted multiple times within 14 Kbp region of DMNA,which is strongly suggestive of a single gene. Digestion with EcoR1,which cuts within the retrovirus, yields different size fragments foreach line, establishing that they represent different insertional eventsand are truly independent isolates. The use of retroviral sequences toclone the genomic DNA flanking the insertion sites should now permitpositive identification of a gene involved in the M2 mechanism.Interference with the function of that gene (for example, usingantisense techniques) should result in the derepression of telomeraseand the ability to extend the lifespan of normal human cells. This geneshould also prove to be mutated in a variety of cancer cells, and isthus likely to be of diagnostic and therapeutic value in cancer as well.

Example 21 Tissue Distribution of Telomerase Activity in Primates

[0399] S100 extracts were prepared from a 12 year old healthy maleRhesus Macaque to determine the tissue distribution of telomeraseactivity. Abundant telomerase activity was detected only from thetestis. Samples of tissue from the brain, kidney, and liver displayed nodetectable activity. This suggests that telomerase inhibition as atherapeutic modality for cancer has the unique advantage of not beingabundant in normal tissues with the exception of the germ line.Therefore telomerase inhibitors should be targeted away from the germcells in reproductive aged individuals to decrease the chance of birthdefects. Such targeting may be accomplished by localized injection orrelease of the active agent near the site of the tumor. The effect ofthe telomerase inhibitors in the male may be easily determined bymeasuring telomere repeat length in the sperm.

1 57 60 nucleic acid single linear 1 TTAGGGTTAG GGTTAGGGTT AGGGTTAGGGTTAGGGTTAG GGTTAGGGTT AGGGTTAGGG 60 12 nucleic acid single linear 2CCCTAACCCT AA 12 12 nucleic acid single linear 3 TTAGGGTTAG GG 12 18nucleic acid single linear 4 CCCTAACCCT AACCCTAA 18 18 nucleic acidsingle linear 5 TTAGGGTTAG GGTTAGGG 18 24 nucleic acid single linear 6CCCTAACCCT AACCCTAACC CTAA 24 12 nucleic acid single linear 7 CCCTAACCCTAA 12 13 nucleic acid single linear 8 ACGGATGTCT AAC 13 10 nucleic acidsingle linear 9 TTCTTGGTGT 10 13 nucleic acid single linear 10ACGGATGTCA CGA 13 10 nucleic acid single linear 11 TCATTGGTGT 10 13nucleic acid single linear 12 AAGGATGTCA CGA 13 13 nucleic acid singlelinear 13 ACGGATGCAG ACT 13 10 nucleic acid single linear 14 CGCTTGGTGT10 25 nucleic acid single linear 15 ACGGATTTGA TTAGTTATGT GGTGT 25 25nucleic acid single linear 16 ACGGATTTGA TTAGGTATGT GGTGT 25 8 nucleicacid single linear 17 CTGGGTGC 8 8 nucleic acid single linear 18TGTGGGGT 8 11 nucleic acid single linear 19 GTGTAAGGAT G 11 5 base pairsnucleic acid single linear 20 TGGTG 5 12 nucleic acid single linear 21ACGGATGTCA CG 12 11 nucleic acid single linear 22 GTGTAAGGAT G 11 26nucleic acid single linear 23 TTAGGGTTAG GGTTAGGGGG GGGGGG 26 26 nucleicacid single linear 24 TTAGGGTTAG GGTTGGGGGG GGGGGG 26 26 nucleic acidsingle linear 25 TTAGGGTTAG GGTGGGGGGG GGGGGG 26 16 nucleic acid singlelinear 26 CCCCCCCCTA ACCCTA 16 16 nucleic acid single linear 27CCCCCCCCAA CCCTAA 16 16 nucleic acid single linear 28 CCCCCCCCAC CCTAAC16 24 nucleic acid single linear 29 TTAGGGTTAG GGTTAGGGTT AGGG 24 37nucleic acid single linear 30 CAATAATGTA TTAAAAATAT GCTACTTATG CATTATC37 23 nucleic acid single linear 31 TGGTACCGTC GAAAGCTTGA CTG 23 14nucleic acid single linear 32 ATGAACTGAC CTAG 14 170 nucleic acid singlelinear 33 GATCTAGGCA CAGCTGCTTC TCATTAGGCA GGTCTCAGCT AGAAGACCACTTCCCTCCCT 60 GAGGAAGTCA ACCCTTCTGC CACCCCATGG CCTTGCTTAA ATTTTCAGACTGTCGAATTG 120 AATCCTACC TCCATTAGCT ACTAGCTTGG GCAAGATACA GAGCCCTCCC 170116 nucleic acid single linear 34 ATATATGCGC TACATAAATG TATCTAGATGCAATTATCTA GATACATATA AGAAAGTATT 60 TGAAGGCCTT CTACAAGGCT TAGTTATTATATTGGTTCAT ACAAGTTCTT CTTCAG 116 176 nucleic acid single linear 35ATCCTTCTCC GCAAACTAAC AGGAACAGAA AACCAAACAC TGCATGTTCT CACATCATTG 60TGGGAGTTGA ACAATGAGAA CACATGGACA CAGGGAGGGG AACATCACAC ACTCGGGGTG 120TCAGCCGGGT GGGAGGGTAG AGGAGGAGAA ATACCTAAGT TCCAGATGAC AGGTTG 176 169nucleic acid single linear 36 GATCTATGCT ACCTCTAGGG ATGGCACCATTCACAAGCAC AAAGGAGATG TCAGTGATTA 60 AAAACACATG CTCTGGAGTC TGAGAGACTTTGAGACTTGC TAGCTTGTGA CTCTGCAGAG 120 TTTAAGGTAT CTGGACCCCT TTTTCCCTCATGTGCATAAT GAAGAGATT 169 192 nucleic acid single linear 37 GATCAACACTGTTAGTTGAG TACCCACATC ACAAACGTGA TTCTCAGAAT GCCTTCCTTC 60 CTGTCTAGTTTCTATAGGTA GATATTTCCT TTTTCAGCAT AGGCCTGAAA AGCCGCCTCC 120 AAATGCCCGCCTTCCAGACA CTATAAAAAG AGGGTTCAAA CCTACTCTAT GAAAGGGAAT 180 GTTCAACACA GA192 157 nucleic acid single linear The letter “N” stands for any base.38 GATCTGTTTA TTATTCTTCC AATATCTCCC CATCTCTTAA AAATTGGTTA TTTCTTCGTT 60CATACATTTT TATCTCCCAA ATTANNNNTG AGACTGGTTT GAAGAGAGGA AAGCAATGTA 120CACACTTTTA TATTCCACCA TGTATATCCG GATATCC 157 135 nucleic acid singlelinear 39 AATCCTCCTA CCTTAACCTC CCTTTGTTAG CCTGCCATTA CAGGTGTGAGCCACCATTGC 60 TCATTCGTCC GTTTATTCAT TCAACAAATC AATCGATCTA TTACATGTGAGGGACTCTTC 120 AGGTCATGGG AATTC 135 117 nucleic acid single linear Theletter “N” stands for any base. 40 GATCACTTGA GCCCAGGAGT TTGAGACCAGCCTGGGTGAC ATGGCAAAAC CCCATCTCTA 60 CCAAAAGAAA AAAANNNNAC AAATTGGTGGTGTTGATGGT CGGCGACCAT TGATCCC 117 137 nucleic acid single linear 41GATCAGGGAG GGGCCGAAAA CTGGAGATGC AGGTGTGCTG TAAGACACTG CAGAGAGGGC 60ATTTACCTGC CCCATCATCC AGCACAGGAA CAGCGACTGA CAGCGCTCAC CCACCCACCA 120TCGCCAGTCA CACTGGG 137 80 nucleic acid single linear 42 GGCTCGAGAACGGGAGGAGG GGGCTCTTGT ATCAGGGCCC GTTGTCACAT CTGCTCTCAG 60 CTTGTTGAAAACTCATAATC 80 157 nucleic acid single linear 43 AGGTCCCTTG GTCGTGATCCGGGAAGGGGC CTGACGTTGC GGGAGATCGA GTTTTCTGTG 60 GGCTTGGGGA ACCTCTCACGTTGCTGTGTC CTGGTGAGCA GCCCGGACCA ATAAACCTGC 120 TTTTCTTAAA AGGAAAAAAAAAAAAAAAAA AAAAAAA 157 120 nucleic acid single linear 44 ATCTAGGTTTTTTAAAAAAG CTTTGAGAGG TAATTACTTG CATATGAGAG AATAAAACAT 60 TTGGCACATTGTTAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 120 149 nucleicacid single linear 45 CTCATTTACT TTTCTCTTAT AGCGTGGCTT TAAACATATATACATTTGTA TATATGTATA 60 TATGAATATA ATGTATAAAA TGTATGTAGA TGTATATACAAAAAATAAAC GAGATGGGTT 120 AAAGATATGT AAAAAAAAAA AAAAAAAAA 149 142nucleic acid single linear 46 AGTCCCAGCT ACTCGGGAGG GCTGAGGCAGGAGAATGGCG TGAACCCAGG AGGCGAAGCT 60 TGCAGTGAGC TGAGATCGCG CCACTGCACTCCAGCCTGGA CGACAGAGCG AGACTCTGTC 120 TCAAAAAAAA AAAAAAAAAA AA 142 7 basepairs nucleic acid single linear 47 TGGTGTG 7 9 base pairs nucleic acidsingle linear 48 TGGTGTGTG 9 11 base pairs nucleic acid single linear 49TGGTGTGTGT G 11 13 base pairs nucleic acid single linear 50 TGGTGTGTGTGTG 13 15 base pairs nucleic acid single linear 51 TGGTGTGTGT GTGTG 15 6base pairs nucleic acid single linear 52 TGGGTG 6 8 base pairs nucleicacid single linear 53 TGGGTGTG 8 10 base pairs nucleic acid singlelinear 54 TGGGTGTGTG 10 12 base pairs nucleic acid single linear 55TGGGTGTGTG TG 12 14 base pairs nucleic acid single linear 56 TGGGTGTGTGTGTG 14 16 base pairs nucleic acid single linear 57 TGGGTGTGTG TGTGTG 16

1. Method for treatment of a condition associated with an elevated levelof telomerase activity within a cell comprising the step of:administering to said cell a therapeutically effective amount of aninhibitor of said telomerase activity.
 2. Method for treatment of acondition associated with an increased rate of proliferation of a cell,comprising the step of: administering to said cell a therapeuticallyeffective amount of an agent active to reduce loss of telomere lengthwithin said cell during said proliferation.
 3. Method for extending theability of a cell to replicate, comprising the step of: administering tosaid cell a replication extending amount of an agent active to reduceloss of telomere length within said cell during cellular replication. 4.A pharmaceutical composition comprising a therapeutically effectiveamount of an inhibitor of telomerase activity in a pharmaceuticallyacceptable buffer.
 5. A pharmaceutical composition comprising atherapeutically effective amount of an agent active to reduce loss oftelomere length within a cell during proliferation of said cell, in apharmaceutically acceptable buffer.
 6. Method for diagnosis of acondition in a patient associated with an elevated level of telomeraseactivity within a cell, comprising the step of: determining the presenceor amount of telomerase within said cells in said patient.
 7. Method fordiagnosis of a condition associated with an increased rate ofproliferation in a cell in an individual, comprising the steps ofdetermining the length of telomeres within said cell.
 8. Method fordetermining telomere length of an animal chromosome or group ofchromosomes, said method comprising: bringing together in a reactionmixture said chromosome(s) or telomere comprising fragment(s) thereof, aprimer having at least two telomeric repeat units, and nucleosidetriphosphates having the same nucleotides as the non-protruding strandof said telomere, wherein at least one of said nucleoside triphosphatesor primer is labeled with a detectable label; and a DNA polymerase;incubating said reaction mixture for sufficient time for said primer tobe extended to provide a primer extended sequence; separating saidprimer extended by size; and determining the size of said primerextended sequence by means of said label.
 9. Method according to claim8, wherein one of nucleoside triphosphates is labeled with aradioisotope and said size is determined by the level of radioactivityin relation to the amount of DNA present.
 10. Method according to claim8, wherein said nucleosides are combinations of A, T and C, or A, T andG.
 11. Method of determining telomere length of an animal chromosome orgroup of chromosomes, said method comprising: fragmenting saidchromosome(s) by a restriction endonuclease having a four baserecognition site absent in the telomere sequence; bringing together saidfragments and a primer for said telomeric sequence, wherein said primeris labeled to allow for binding of said primer to a surface;cross-linking said primer to said telomeric sequence; isolating saidtelomeric sequence by means of said label; and determining the size ofsaid telomeric sequence bound to said surface.
 12. Method according toclaim 11, wherein said primer is conjugated with (1) an agent capable ofcross-linking nucleic acids upon irradiation; and (2) a specific bindingpair member; and said surface is conjugated with the complementaryspecific binding pair member.
 13. Method according to claim 11, whereinsaid primer is conjugated with (1) an agent capable of cross-linkingnucleic acids upon irradiation; and (2) a particle.
 14. Method ofreducing the rate of telomere shortening in a proliferating cellularcomposition, said method comprising: introducing into cells of saidcellular composition primers having from 2 to 3 repeats of the repeatingunit of the cellular telomere.
 15. Method of measuring the telomeraseactivity of a composition, said method comprising: combining 1 or morerepeats of the telomere unit sequence and nucleoside triphosphateslacking cytidine nucleotide, wherein at least one of said primer ornucleoside triphosphates is labeled with a detectable label, with theproviso that when said composition lacks a telomere sequencecomplementary to said probe, said telomere sequence is added to saidcomposition; incubating said composition for a predetermined time forsaid primer to be extended to provide an extended sequence; anddetermining the rate of formation of said extended sequence.
 16. Methodaccording to claim 15, wherein one of said nucleoside triphosphates islabeled with a radioisotope, and said determining is by measuringradioactivity per unit weight of DNA.
 17. Method of inhibiting theproliferation of telomerase-comprising immortalized cells, said methodcomprising: contacting said immortalized cells with a telomeraseinhibitor under conditions wherein said inhibitor enters said cells;whereby said proliferation of said cells is inhibited.
 18. Methodaccording to claim 17, wherein said inhibitor inhibits expression oftelomerase.
 19. Method of according to claim 17, wherein said inhibitionis an oligonucleotide sequence comprising the complementary sequence ofthe telomerase RNA.
 20. Method according to claim 17, wherein saidoligonucleotide sequence is a ribozyme.
 21. Method for extending theproliferative capability of a mammalian cell population, said methodcomprising: adding to said cells oligonucleotides comprising at leasttwo repeats complementary to the sequence of the protruding strand ofthe telomere of the chromosomes of said cells, whereby the shortening ofsaid telomere is slowed.
 22. Method for treatment of a disease orcondition associated with cell senescense, comprising the steps of:administering a therapeutically effective amount of an agent active toderepress telomerase in the senescing cells.
 23. Method for screeningfor a telomerase derepression agent, comprising the steps of: contactinga potential agent with a cell lacking telomerase activity, anddetermining whether said agent increases the level of said activity. 24.The method of claim 23, wherein said cell is a cell expressing aninducible T antigen.
 25. The method of claim 1, wherein said cell if afungal cell, and said administering reduces viability of said cell. 26.The method of claim 25, wherein said cell is a C. albicans cell. 27.Method for screening for agents useful in treatment of a human diseaseassociated with an elevated level of telomerase activity in a humancell, comprising the step of testing potential said agents for activityin inhibiting telomerase activity.